Cyclic peptide tyrosine tyrosine compounds as modulators of neuropeptide y receptors

ABSTRACT

The present invention comprises compounds of Formula I. 
     
       
         
         
             
             
         
       
     
     wherein:
     Z 4 , Z 7 , Z 9 , Z 11 , Z 22 , Z 23 , Z 26 , Z 30 , Z 34 , Z 35 , p, m, n, q, and BRIDGE are defined in the specification. The invention also relates to pharmaceutical compositions and methods for use thereof. The novel compounds are useful for preventing, treating or ameliorating diseases and disorders, such as obesity, type 2 diabetes, the metabolic syndrome, insulin resistance, and dyslipidemia, among others.

FIELD OF THE INVENTION

The present invention is directed generally to novel cyclic peptidetyrosine tyrosine (PYY) compounds, which are modulators of theneuropeptide Y2 receptor. The invention also relates to pharmaceuticalcompositions and methods for use thereof. The novel compounds are usefulfor preventing, treating or ameliorating diseases and disorders, such asobesity, type 2 diabetes, the metabolic syndrome, insulin resistance,and dyslipidemia, among others.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/413,613, filed on Oct. 27, 2016, and U.S. Provisional PatentApplication No. 62/413,586 filed on Oct. 27, 2016. Each disclosure isincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “PRD3411 Sequence Listing” and a creation date of Oct. 23,2017, and having a size of 96 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety. In the event of any inconsistency with regardto the structures for SEQ ID NOs: 1-111 between the informationdescribed herein and the Sequence Listing submitted electronically viaEFS-Web with a file name “PRD3411 Sequence Listing,” the informationherein will prevail.

BACKGROUND OF THE INVENTION

Neuropeptide Y (NPY) receptors are activated by a closely related groupof peptide agonists termed “NPY family” which have differing affinitiesfor each receptor sub-type. NPY, peptide tyrosine-tyrosine (PYY) andpancreatic polypeptide (PP), all 36 amino acids in length, are agonistsfor the NPY family of receptors. NPY is a neurotransmitter, synthesized,co-stored and released with norepinephrine and epinephrine. NPY is oneof the most abundant and widely distributed peptides in the centralnervous system (CNS) of humans and rodents and is expressed in areas ofthe brain related to feeding and stress. In the peripheral nervoussystem, NPY-containing neurons are predominantly sympathetic. PYY ispredominantly synthesized and released by intestinal endocrine cells.Cleavage of NPY and PYY by the endothelial serine-protease, di-peptidylpeptidase IV (DPP-IV), generates NPY₃₋₃₆ and PYY₃₋₃₆ which are selectiveligands for Y2 and Y5 sub-types of the NPY receptor family. PP is mainlyfound in pancreatic islet cells distinct from those storing insulin,glucagon or somatostatin.

Five distinct NPY receptors have been identified to date, four of whichare understood as relevant to human physiology. The receptors Y1, Y2 andY5 preferentially bind NPY and PYY, whereas the Y4 receptorpreferentially binds PP. The Y2 and Y5 receptors are also potentlyactivated by NPY3-36 and PYY3-36. In general, the NPY family of ligandspossesses variable selectivity for each of the NPY receptor isoforms,with PYY3-36 previously reported to have modest-to-robust selectivityfor the Y2 isoform. Each of these receptors is coupled to inhibition ofadenylate cyclase via pertussis-toxin sensitive Gαi.

PYY is secreted from endocrine L-cells in response to food, and inparticular following fat ingestion. PYY₁₋₃₆ predominates in the fastingstate, with PYY₃₋₃₆ being the major form found post-prandially inhumans, with plasma concentrations negatively correlated with the numberof calories consumed. PYY3-36 has been demonstrated to reduce foodintake in humans, monkeys, rats, rabbits, and mice (Batterham R L et al.Nature 2002 Aug. 8; 418(6898):650-4; Batterham R L et al. N Engl J Med2003 Sep. 4; 349(10):941-8; Challis B G et al., Biochem Biophys ResCommun 2003 Nov. 28; 311(4):915-9). The anorexigenic effects of PYY₃₋₃₆are believed to be Y2-mediated, based on preferential binding at thisreceptor and loss of feeding efficacy in Y2-deficient mice (Batterham RL, et al. Nature 2002 Aug. 8; 418(6898):650-4). Intra-arcuate injectionof PYY3-36 reduces food intake in rats and mice (Batterham et al. Nature2002 Aug. 8; 418(6898):650-4), suggesting that engagement ofhypothalamic Y2 receptors may mediate these effects. Acute effects onfeeding have also been shown to translate to dose-dependent effects onbody-weight in ob/ob mice, DIO mice and Zucker fa/fa mice (Pittner R Aet al. Int J Obes relat Metab Disord 2004 August; 28(8):963-71). Inaddition, PYY₃₋₃₆ has also been shown to improve insulin-mediatedglucose disposal and insulin sensitivity in DIO rodents (Vrang N et al.,Am J Physiol Regul Integr Comp Physiol August; 291(2):R367-75).Bariatric surgery results in increased circulating PYY-immunoreactivity(le Roux C W et al., Ann Surg 2006 January; 243(1); 108-14), whichappears to play a role in postoperative weight loss.

Given its role in controlling appetite and food intake as well as itsanti-secretory and pro-absorptive effects in the gastrointestinal tractin mammals, PYY₃₋₃₆ may be effective in treating obesity and associatedconditions as well as in a number of gastrointestinal disorders.However, the therapeutic utility of PYY₃₋₃₆ itself as a treatment agentis limited by its rapid metabolism and resultant short circulatinghalf-life (Torang et al., Am. J. Physiol. Regul. Integr. Comp. Physiol.310:R866-R874 (2016)).

Thus, it is desirable to obtain a PYY analogue or derivative thereofwith an improved metabolic stability and pharmacokinetic profilerelative to PYY3-36. Such derivatives, with a protracted half-life invivo, would provide Y2 receptor modulation with greater duration ofaction, making them suitable as therapeutic agents for subjects in needof such modulation.

The foregoing discussion is presented solely to provide a betterunderstanding of the nature of the problems confronting the art andshould not be construed in any way as an admission as to prior art norshould the citation of any reference herein be construed as an admissionthat such reference constitutes “prior art” to the instant application.

SUMMARY OF THE INVENTION

One general aspect of the invention relates to a compound of Formula I:

whereinp is 0 or 1;m is 0, 1, 2, 3, 4, or 5;n is 1, 2, 3, or 4;q is 0 or 1; provided that q is 1 only when Z₃₀ is absent;BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —SCH₂C(O)NH—,—(OCH₂CH₂)₂NHC(O)CH₂S, —NHC(O)—, or —CH₂S—;

Z₄ is K, A, E, S, or R; Z₇ is A or K; Z₉ is G or K; Z₁₁ is D or K; Z₂₂is A or K; Z₂₃ is S or K; Z₂₆ is A or H;

Z₃₀ is L, W, absent, or K;provided that Z₃₀ is absent only when q is 1;

Z₃₄ is

Z₃₅ is

or a derivative thereof; wherein the derivative is the compound ofFormula I that is modified by one or more processes comprisingamidation, glycosylation, carbamylation, sulfation, phosphorylation,cyclization, lipidation, or pegylation; or a pharmaceutically acceptablesalt thereof.

The present invention also provides methods of preventing, treating,delaying the onset of, or ameliorating a syndrome, disorder or disease,or any one or more symptoms of said syndrome, disorder, or disease,wherein said syndrome, disorder or disease is selected from the groupconsisting of obesity, type 2 diabetes, metabolic syndrome (i.e.,Syndrome X), insulin resistance, impaired glucose tolerance (e.g.,glucose intolerance), hyperglycemia, hyperinsulinemia,hypertriglyceridemia, dyslipidemia, atherosclerosis, diabeticnephropathy, and other cardiovascular risk factors such as hypertensionand cardiovascular risk factors related to unmanaged cholesterol and/orlipid levels, osteoporosis, inflammation, and eczema, comprisingadministering to a subject in need thereof an effective amount of acompound of Formula I, a derivative or pharmaceutically acceptable saltthereof, or a form, composition or medicament thereof, or any of thecombinations described herein.

The present invention also contemplates preventing, treating, delayingthe onset of, or ameliorating any of the diseases, disorders, syndromes,or symptoms described herein with a combination therapy that comprisesadministering to a subject in need thereof an effective amount of acompound of Formula I, a derivative or pharmaceutically acceptable saltthereof, or a form, composition or medicament thereof, in combinationwith any one or more of the following additional compounds: a dipeptidylpeptidase-4 (DPP-4) inhibitor (e.g., sitagliptin, saxagliptin,linagliptin, alogliptin, etc.); a GLP-1 receptor agonist (e.g.,short-acting GLP-1 receptor agonists such as exenatide and lixisenatide;intermediate-acting GLP-1 receptor agonists such as liraglutide;long-acting GLP-1 receptor agonists such as exenatide extended-release,albiglutide, dulaglutide); a sodium-glucose co-transporter-2 (SGLT-2)inhibitors (e.g., canaglifozin, dapaglifozin, empaglifozin, etc.); bileacid sequestrants (e.g., colesevelam, etc.); and dopamine receptoragonists (e.g., bromocriptine quick-release). In some embodiments, thedose of the additional compound is reduced when given in combinationwith a compound of Formula I, a derivative or pharmaceuticallyacceptable salt thereof. In some embodiments, when used in combinationwith a compound of Formula I, the additional compounds may be used inlower doses than when each is used singly.

The present invention contemplates preventing, treating, delaying theonset of, or ameliorating any of the diseases, disorders, syndromes, orsymptoms described herein with a combination therapy that comprisesadministering to a subject in need thereof an effective amount of acompound of Formula I, a derivative or pharmaceutically acceptable saltthereof, or a form, composition or medicament thereof, in combinationwith any one or more of the following additional compounds: biguanides(e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g.,chlorpropamide, glimepiride, glipizide, glyburide, glibenclamide,glibornuride, glisoxepide, glyclopyramide, tolazamide, tolbutamide,acetohexamide, carbutamide, etc.); and thiazolidinediones (e.g;pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone,englitazone, netoglitazone, rivoglitazone, troglitazone, etc.). In someembodiments, when used in combination with a compound of Formula I, aderivative or pharmaceutically acceptable salt thereof, the additionalcompounds may be used in lower doses than when each is used singly.

In yet other embodiments, the present invention contemplates preventing,treating, delaying the onset of, or ameliorating any of the diseases,disorders, syndromes, or symptoms described herein, with a combinationtherapy that comprises administering to a subject in need thereof aneffective amount of a compound of Formula I, a derivative orpharmaceutically acceptable salt thereof, or a form, composition ormedicament thereof, in combination with a surgical therapy, such asbariatric surgery (e.g., gastric bypass surgery, such as Roux-en-Ygastric bypass surgery; sleeve gastrectomy; adjustable gastric bandsurgery; biliopancreatic diversion with duodenal switch; intragastricballoon; gastric plication; and combinations thereof).

Further aspects, features and advantages of the present invention willbe better appreciated upon a reading of the following detaileddescription of the invention and claims.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences (e.g., cyclic PYY₃₋₃₆polypeptide sequences), refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection using methods known in theart in view of the present disclosure.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human. The term “mammal” as used herein, encompasses anymammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans, etc., more preferably a human.

The term “administering” with respect to the methods of the invention,means a method for therapeutically or prophylactically preventing,treating or ameliorating a syndrome, disorder or disease as describedherein by using a compound of the invention or a pharmaceuticallyacceptable salt thereof, optionally conjugated to a half-life extensionmoiety, or a form, composition or medicament thereof. Such methodsinclude administering an effective amount of said compound, compoundform, composition or medicament at different times during the course ofa therapy or concurrently in a combination form. The methods of theinvention are to be understood as embracing all known therapeutictreatment regimens.

The term “effective amount” means that amount of active compound orpharmaceutical agent that elicits the biological or medicinal responsein a tissue system, animal or human, that is being sought by aresearcher, veterinarian, medical doctor, or other clinician, whichincludes preventing, treating or ameliorating a syndrome, disorder, ordisease being treated, or the symptoms of a syndrome, disorder ordisease being treated.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

Compounds

In one general aspect, the present invention comprises a compound ofFormula I:

whereinp is 0 or 1;m is 0, 1, 2, 3, 4, or 5;n is 1, 2, 3, or 4;q is 0 or 1; provided that q is 1 only when Z₃₀ is absent;BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —SCH₂C(O)NH—,(OCH₂CH₂)₂NHC(O)CH₂S, —NHC(O)—, or —CH₂S—;

Z₄ is K, A, E, S, or R; Z₇ is A or K; Z₉ is G or K; Z₁₁ is D or K; Z₂₂is A or K; Z₂₃ is S or K; Z₂₆ is A or H;

Z₃₀ is L, W, absent, or K;provided that Z₃₀ is absent only when q is 1;

Z₃₄ is

Z₃₅ is

or a derivative thereof; wherein the derivative is the compound ofFormula I that is modified by one or more processes comprisingamidation, glycosylation, carbamylation, sulfation, phosphorylation,cyclization, lipidation, or pegylation; or a pharmaceutically acceptablesalt thereof.

In one embodiment, the invention comprises the compound of claim 1 or aderivative thereof, wherein the derivative is the compound of Formula Ithat is modified by one or more processes comprising amidation,lipidation, or pegylation; or a pharmaceutically acceptable saltthereof.

In another embodiment, the invention comprises the compound of Formula Ior a derivative thereof, wherein:

p is 0 or 1;m is 0, 1, 2, 3, 4, or 5;n is 1, 2, 3, or 4;q is 0 or 1; provided that q is 1 only when Z₃₀ is absent;BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —SCH2C(O)NH—,—(OCH₂CH₂)₂NHC(O)CH₂S, —NHC(O)—, or —CH₂S—;

Z₄ is K, A, E, S, or R;

Z₇ is A or K, wherein the amino side chain of said K is optionallysubstituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl;Z₉ is G or K, wherein the amino side chain of said K is optionallysubstituted with

wherein t is 0, 1, or 2;

u is 0 or 1; and

v is 14, 16, or 18;

x wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl;Z₁₁ is D or K, wherein the amino side chain of said K is optionallysubstituted with

wherein w is 0, 1, 2, or 4;

x is 0 or 1; and

y is 14, 16, or 18;

x, wherein i is an integer of 0 to 24, and X═Br, I or Cl

Z₂₂ is A or K, wherein the amino side chain of said K is optionallysubstituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl;Z₂₃ is S or K, wherein the amino side chain of said K is optionallysubstituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl

Z₂₆ is A or H;

Z₃₀ is L, W, absent, or K, provided that Z₃₀ is absent only when q is 1,wherein the amino side chain of said K is optionally substituted with

wherein r is 0, 1, or 2;

s is 0 or 1; and

q is 14, 16, or 18; or

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention comprises the compound of Formula Ior a derivative thereof, wherein:

p is 0 or 1;m is 0, 1, 2, 3, or 5;n is 1, 2, or 4;q is 0 or 1; provided that q is 1 only when Z₃₀ is absent;BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —(OCH₂CH₂)₂NHC(O)CH₂S,—NHC(O)—, or —CH₂S—;

Z₄ is K, A, E, S, or R;

Z₇ is A or K, wherein the amino side chain of said K is substituted with

Z₉ is G or K, wherein the amino side chain of said K is substituted with

wherein t is 0;

u is 1; and

v is 14;

Z₁₁ is D or K, wherein the amino side chain of said K is optionallysubstituted with

wherein w is 0, or 4;

x is 1; and

y is 14;

Z₂₂ is A or K, wherein the amino side chain of said K is substitutedwith

Z₂₃ is S or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L, W, absent, or K, provided that Z₃₀ is absent only when q is 1,wherein the amino side chain of said K is substituted with

wherein r is 0, or 2;

s is 1; and

q is 14, 16, or 18; or

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention comprises the compound of Formula Ior a derivative thereof, wherein:

p is 0 or 1;m is 0, 1, 2, 3, or 5;n is 1, 2, or 4;q is 0 or 1; provided that q is 1 only when Z₃₀ is absent;BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —(OCH₂CH₂)₂NHC(O)CH₂S,—NHC(O)—, or —CH₂S—;

Z₄ is K, A, E, S, or R;

Z₇ is A or K, wherein the amino side chain of said K is substituted with

Z₉ is G or K, wherein the amino side chain of said K is substituted with

wherein t is 0;

u is 1; and

v is 14;

Z₁₁ is D or K, wherein the amino side chain of said K is optionallysubstituted with

wherein w is 0, or 4;

x is 1; and

y is 14;

Z₂₂ is A or K, wherein the amino side chain of said K is substitutedwith

Z₂₃ is S or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L, W, absent, or K, provided that Z₃₀ is absent only when q is 1,wherein the amino side chain of said K is substituted with

wherein r is 0, or 2;

s is 1; and

q is 14, 16, or 18; or

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

Another embodiment of the invention is a compound of Formula I or aderivative thereof, selected from the group consisting of SEQ ID NO: 1to SEQ ID NO: 110, or a pharmaceutically acceptable salt thereof.

Another embodiment of the invention is a compound of Formula I or aderivative thereof, selected from the group consisting of SEQ ID NO: 2to SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof.

Another general aspect of the invention relates to a conjugatecomprising a compound of Formula I, a derivative or a pharmaceuticallyacceptable salt thereof and a half-life extension moiety conjugatedthereto. As used herein, the term “conjugated” refers to a compound ofthe invention covalently linked to or covalently connected to ahalf-life extension moiety, directly or via a linker. In the presentdisclosure, with respect to a compound of Formula I, a derivative or apharmaceutically acceptable salt thereof, the phrase “a conjugatecomprising a compound and a half-life extension moiety conjugatedthereto” is used interchangeably with the phrase “a compound conjugatedto a half-life extension moiety.”

As used herein, the term “linker” refers to a chemical module comprisinga covalent or atomic chain that covalently connects a compound of theinvention to a half-life extension moiety. The linker can, for example,include, but is not limited to, a peptide linker, a hydrocarbon linker,a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker,a polysaccharide linker, a polyester linker, a hybrid linker consistingof PEG and an embedded heterocycle, and a hydrocarbon chain. The linkercan, for example, be first covalently connected to a compound of theinvention, then covalently connected to a half-life extension moiety.

As used herein, a “half-life extension moiety” is used interchangeablywith the term “half-life extending moiety.” Exemplary half-lifeextension moieties include, but are not limited to, monoclonalantibodies or fragments thereof, albumin, albumin variants,albumin-binding proteins and/or domains, transferrin and fragments andanalogues thereof. Additional half-life extension moieties that can beincorporated into the conjugates of the invention include, for example,polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000,α-tocopherolyl, fatty acids and fatty acid esters of different chainlengths, for example laurate, myristate, stearate, arachidate, behenate,oleate, arachidonate, octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like, polylysine,octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides)for desired properties.

The compounds of the invention can be covalently linked to one or moreof half-life extension moieties using methods known in the art in viewof the present disclosure. For example, as illustrated by the Examplesbelow, a half-life extension moiety, such as a PEG moiety or alipophilic moiety, can be added to a peptide molecule of the invention,e.g., by incorporating a cysteine or lysine residue to the molecule andattaching the half-life extension moiety to the cysteine or lysine usingwell known methods. Examples of compounds of the invention conjugated toa monoclonal antibody as the half-life extension moiety are alsodescribed in U.S. Provisional Patent Application No. 62/413,586, filedon Oct. 27, 2016, and U.S. patent application Ser. No. ______ entitled“Antibody-coupled cyclic peptide tyrosine tyrosine compounds asmodulators of neuropeptide receptors,” filed on the same day as thisapplication with the Attorney Docket Number PRD3436, the contents ofboth applications are hereby incorporated by reference in theirentireties.

According to embodiments of the invention, an electrophile is introducedonto a sidechain of a cyclic PYY of the invention, such asbromoacetamide or maleimide, that reacts site specifically with thesulfhydryl group of the Cys residue engineered into a half-lifeextension moiety, such as a monoclonal antibody or fragment thereof,thereby creating a covalent linkage between the cyclic PYY peptide andthe half-life extension moiety. A compound of the invention can becovalently linked to one or more of half-life extension moietiesdirectly, or through a linker. Linkers useful for the invention include,but are not limited to, a peptide linker, a hydrocarbon linker, apolyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, apolysaccharide linker, a polyester linker, or a hybrid linker consistingof PEG and an embedded heterocycle. In certain embodiments, thehalf-life extension moiety with a linker, can be conjugated to acompound of the invention at one or more amino acid positions of thecyclic PYY, such as amino acid residue 4, 7, 9, 10, 11, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 26, 30, or 31 of the PYY using methodsknown in the art. In certain embodiments, the half-life extension moietywithout a linker, can be conjugated to a compound of the invention atone or more amino acid positions of the cyclic PYY, such as amino acidresidue 4, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,26, 30, or 31 of the PYY using methods known in the art. The amino acidresidue numbering follows that of hPYY₃₋₃₆. Any of the compounds of thepresent invention, including but not limited to SEQ ID NO: 1 to SEQ IDNO: 110 can be conjugated to a half-life extension moiety, directly orindirectly through a linker. According to embodiments of the invention,a compound selected from the group consisting of SEQ ID NO: 74, 95, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110, or apharmaceutical acceptable salt thereof, can be covalently linked to ahalf-life extension moiety, such as a monoclonal antibody or a fragmentthereof, via a linker.

A peptide molecule of the invention, or a conjugate comprising thepeptide molecule covalently linked to one or more half-life extensionmoieties can be assayed for functionality by known assays in view of thepresent disclosure. For example, the biological or pharmacokineticactivities of a peptide molecule of the invention, alone or in aconjugate according to the invention, can be assayed using known invitro or in vivo assays and compared.

In one embodiment, the present invention comprises a compound of FormulaII

whereinp is 0 or 1;m is 0, 1, 2, 3, 4, or 5;n is 1, 2, 3, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S;

Z₉ is G or K, wherein the amino side chain of said K is substituted with

wherein t is 0, 1, or 2;

u is 0 or 1; and

v is 14, 16, or 18;

Z₁₁ is D or K, wherein the amino side chain of said K is substitutedwith

wherein w is 0, 1, or 2;

x is 0 or 1; and

y is 14, 16, or 18;

Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein r is 0, 1, or 2;

s is 0 or 1; and

q is 14, 16, or 18; or

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II, wherein:

p is 0 or 1;m is 0, 2, 3, or 5;n is 1, 2, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S;

Z₉ is G or K, wherein the amino side chain of said K is substituted with

Z₁₁ is D or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein r is 0, or 2;

s is 0 or 1; and

q is 14, 16, or 18; Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II,

whereinp is 0 or 1;m is 0, 2, 3, or 5;n is 1, 2, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S;

Z₉ is G or K, wherein the amino side chain of said K is substitutedwith

Z₁₁ is D or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein r is 0, or 2;

s is 0 or 1; and

q is 14, 16, or 18;

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II, wherein:

p is 0 or 1;m is 0, 2, 3, or 5;n is 1, 2, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S;

Z₉ is G or K, wherein the amino side chain of said K is substitutedwith

Z₁₁ is D or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein q is 14, 16, or 18; Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II, wherein:

p is 0 or 1;m is 0, 2, 3, or 5;n is 1, 2, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S;

Z₉ is G or K, wherein the amino side chain of said K is substitutedwith

Z₁₁ is D or K, wherein the amino side chain of said K is substitutedwith

Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein q is 14, 16, or 18;

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II, wherein:

p is 0 or 1;m is 0, 2, 3, or 5;n is 1, 2, or 4;BRIDGE is -Ph-CH₂—S—, -triazolyl-, or —NHC(O)CH₂S—;

Z₄ is K, A, E, or S; Z₉ is G; Z₁₁ is D; Z₂₆ is A or H;

Z₃₀ is L or K, wherein the amino side chain of said K is substitutedwith

wherein q is 14, 16, or 18;

Z₃₄ is

Z₃₅ is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention comprises a compound ofFormula II selected from the group consisting of SEQ ID NO: 2 to SEQ IDNO: 46.

Another embodiment of the invention comprises N-terminus to side chaincyclic analogues of PYY exhibiting at least 70%, 75% 80%, 85%, 90%, 95%,or 99% sequence identity to hPYY(₃₋₃₆). As an example of a method fordetermination of the sequence identity between two analogues the twopeptides

are aligned. The sequence identity of the analogue relative tohPYY(₃₋₃₆) is given by the total number of aligned residues minus thenumber of different residues (i.e. the number of aligned identicalresidues) divided by the total number of residues in hPYY₃₋₃₆. In thisexample the different residues are D11 which has been exchanged for asubstituted K11, followed by V31 which has been exchanged for hC31, andfinally R35 has been decarbonylated. Accordingly, in said example thesequence identity is (34−3)/34×100.

Cyclic PYY Peptides

PYY₃₋₃₆ is an endogenous hormone secreted by L cells in the distal gutthat acts as an agonist of the Y2 receptor to inhibit food intake. Givenits role in controlling appetite and food intake as well as itsanti-secretory and pro-absorptive effects in the gastrointestinal tractin mammals, PYY₃₋₃₆ may be effective in treating obesity and associatedconditions as well as in a number of gastrointestinal disorders.However, the therapeutic utility of PYY₃₋₃₆ itself as a treatment agentis limited by its rapid metabolism and short circulating half-life.Thus, the present invention is generally directed to modified PYY₃₋₃₆conjugates, which extend the half-life of the PYY3-36 peptide andreduces the metabolism of the peptide in vivo.

In certain embodiments of the invention, the modified PYY₃₋₃₆ peptidesare cyclic PYY peptides. The terms “cyclic PYY peptide,” “cyclic PYY₃₋₃₆analog,” and “cyclic PYY₃₋₃₆ peptide analog” can be usedinterchangeably.

As used herein, the term “NTSC-PYY” is intended to describeN-terminus-to-side-chain cyclic analogues of PYY.

The peptide sequences described herein are written according to theusual convention whereby the N-terminal region of the peptide is on theleft and the C-terminal region is on the right. Although isomeric formsof the amino acids are known, it is the L-form of the amino acid that isrepresented unless otherwise expressly indicated. For convenience indescribing the molecules of this invention, conventional andnon-conventional abbreviations for various amino acids (both single andthree-letter codes) and functional moieties are used. Theseabbreviations are familiar to those skilled in the art, but for clarityare listed as follows: A=Ala=alanine; R=Arg=arginine; N=Asn=asparagine;D=Asp=aspartic acid; βA=βAla=beta-alanine; C=Cys=cysteine;hC=hCys=homocysteine; E=Glu=glutamic acid; Q=Gln=glutamine;G=Gly=glycine; H=His=histidine; I=Ile=isoleucine; L=Leu=leucine;K=Lys=lysine; Nle=norleucine; F=Phe=phenylalanine; P=Pro=proline;S=Ser=serine; T=Thr=threonine; W=Trp=tryptophan; Y=Tyr=tyrosine andV=Val=valine.

For convenience, the amino acid residue numbering convention used innaming the NTSC-PYY peptides of the present invention follows that ofhPYY₃₋₃₆. Specific amino acid replacements that have been introducedinto the NTSC-PYY peptides, relative to the native residues at thecorresponding positions in hPYY₃₋₃₆, are indicated by the appropriateamino acid code, followed by the position of the substitution. Thus,“S4” in the NTSC-PYY peptide refers to a peptide in which serine hasreplaced the corresponding native lys4 residue of hPYY₃₋₃₆. Similarly,“hC31” in the NTSC-PYY peptide refers to a peptide in which homocysteinehas replaced the corresponding native val31 residue of hPYY₃₋₃₆.Additional amino acid replacements occurring within NTSC-PYY peptidesare described according to this convention and will be recognized assuch by one skilled in the art.

Also for convenience, the naming convention used for the NTSC-PYYpeptides of the present invention incorporates the amino residuesinvolved in the cycle along with the linking group(s) between them in aleft-to-right direction, starting from the N-terminal residue involvedin the cycle. In all cases, the N-terminal amino acid residue of thecycle links by way of its α-amino functionality to the linking group,which in turn connects to the side chain residue of the amino acid atposition 31 of the NTSC-PYY peptide. Thus, “cyclo-(I3-m-COPhCH₂-hC31)”is used to describe the cycle of an NTSC-PYY peptide in which theα-amino functionality of Ile3 is acylated with a meta-toluic acidresidue, whose methyl group is further linked by way of a thioether bondto the side chain of a hCys31 residue. Similarly,“cyclo-(K4-CO(CH₂)₂NHCOCH₂-hC31)” is used to describe the cycle of anNTSC-PYY peptide, in which the native Ile3 residue has been deleted andwhose (now N-terminal) α-amino functionality of lys4 is acylated by a3-acetamidopropanoyl group, whose acetamido methylene carbon isconnected to the side chain of a hCys31 residue by way of a thioetherbond.

Lysine residues can be incorporated at various positions of the hPYY₃₋₃₆sequence to provide a convenient functional handle for furtherderivatization. The lysine residues can be modified to be coupled to themonoclonal antibody either directly or indirectly. In an indirectcoupling to the monoclonal antibody, the lysine residue can be modifiedto comprise a linker which will allow for the cyclic PYY peptide to becoupled to the monoclonal antibody. One skilled in the art willrecognize that related orthologues could also be effectively employed assuch and are contemplated herein.

The term, “K(γ-Glu)”, appearing in the peptide sequence, represents alysinyl residue whose side chain ε-amino group has been acylated by theγ-carboxyl group of glutamic acid.

The term, “K(γ-Glu-Pal (palmitoyl))” represents a lysinyl residue whoseside chain g-amino group has been acylated by the γ-carboxyl group ofN-hexadecan-1-oylglutamic acid.

The term, “K(γ-Glu-Stear (stearoyl))” represents a lysinyl residue whoseside chain g-amino group has been acylated by the γ-carboxyl group ofN-octadecan-1-oylglutamic acid.

The term, “K(γ-Glu-Arach (arachidoyl))” represents a lysinyl residuewhose side chain g-amino group has been acylated by the γ-carboxyl groupof N-dodecan-1-oylglutamic acid.

The term, “K(OEG) (8-amino-3,6-dioxaoctanoyl)” represents a lysinylresidue whose side chain ε-amino group has been acylated by8-amino-3,6-dioxaoctanoic acid.

The term, “(OEG)₂” represents two OEG units linked together insuccession via an amide linkage (i.e.,17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid).

The term, “K(OEG)₂” represents a lysinyl residue whose side chainε-amino group has been acylated by17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid.

The term, “K((OEG)₂-γ-Glu” represents a lysinyl residue whose side chainε-amino group has been acylated by(22S)-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioicacid via its 1-carboxylic acid functionality.

The term, “K((OEG)₂-γ-Glu-Stear)” represents a lysinyl residue whoseside chain g-amino group has been acylated by(22S)-10,19-dioxo-22-stearamido-3,6,12,15-tetraoxa-9,18-diazatricosanedioicacid via its 1-carboxylic acid functionality.

The term, “K((OEG)₂-γ-Glu-COC₁₆CO₂H)” represents a lysinyl residue whoseside chain ε-amino group has been acylated by(21S)-9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonatriacontane-1,21,39-tricarboxylicacid via its 1-carboxylic acid functionality.

Similarly, the term, “K((OEG)₂-γ-Glu-COC₁₈CO₂H)” represents a lysinylresidue whose side chain ε-amino group has been acylated by(21S)-9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazahentetracontane-1,21,41-tricarboxylicacid via its 1-carboxylic acid functionality.

The term, “K((OEG)₂-COC₁₆CO₂H)” represents a lysinyl residue whose sidechain g-amino group has been acylated by10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazahexatriacontanedioic acid viaits 1-carboxylic acid functionality.

The term “K(PEG24-AcBr)” represents a lysinyl residue whose side chainε-amino group has been acylated byN-bromoacetyl-75-amino-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxapentaheptacontanoicacid via its 1-carboxylic acid functionality.

The term “K(PEG12-AcBr)” represents a lysinyl residue whose side chainε-amino group has been acylated byN-bromoacetyl-39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxanonatriacontanoicacid via its 1-carboxylic acid functionality.

The term “K(PEG6-AcBr)” represents a lysinyl residue whose side chainε-amino group has been acylated byN-bromoacetyl-3-[(17-amino-3,6,9,12,15-pentaoxaheptadec-1-yl)oxy]-propanoicacid via its 1-carboxylic acid functionality.

The term “K(PEG8-triazolyl-CH₂CH₂CO—PEG4-AcBr)” represents a lysinylresidue whose side chain ε-amino group has been acylated by27-[4-[2-[3-[2-[2-[3-(N-bromoacetylamino)propoxy]ethoxy]ethoxy]propylaminocarbonyl]ethyl]tetrazol-1-yl]-4,7,10,13,16,19,22,25-octaoxaheptacosanoicacid via its 1-carboxylic acid functionality.

The term “K(mPEG16) represents a lysinyl residue whose side chainε-amino group has been acylated by4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49-hexadecaoxapentacontanoicacid via its 1-carboxylic acid functionality.

The term “K(mPEG12)” represents a lysinyl residue whose side chainε-amino group has been acylated by4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxaoctatriacontanoic acid viaits 1-carboxylic acid functionality.

The term, “VitE” represents an α-tocopherolyl unit in the molecule.

The term, “AcVitE” represents an α-tocopherolyl unit whose phenolicgroup bears an ether-linked methylenylcarboxy functionality.

The term, “K-γ-Glu-AcVitE” represents a lysinyl residue whose side chainε-amino group has been acylated by(2-(((2R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)oxy)acetyl)-L-glutamicacid via its γ-carboxylic acid functionality.

Many of the compounds of the present invention incorporate a reducedamide bond between the C-terminal residue of the sequence, Y36, and itsadjacent residue, R35. This reduced amide linkage is represented by theterm, “psi-(R35,Y36)”.

Various amino acid residues comprising certain sequences of the presentinvention contain α-amino groups that have been methylated. Thus, theterms, “N-Me-Q34” or “N-Me-R35” represent α-N-methylated glutamine atposition 34 of a sequence, and α-N-methylated arginine at position 35 ofa sequence, respectively.

The term, “N-Me-Q34, psi-(R35,Y36)” in a sequence description refers toa sequence containing both an α-methyl glutamine residue at position 34,as well as a reduced amide bond between residues R35 and Y36.

Similarly, the term, “N-Me-R35, psi-(R35,Y36)” in a sequence descriptionrefers to a sequence containing both an α-methyl arginine residue atposition 35, as well as a reduced amide bond between this residue andY36.

As used herein, the term “pegylation” refers to covalent conjugates ofone or more polyethylene glycol (PEG) molecules and one or more NTSC-PYYpeptides. Said conjugates may include, but are not limited to, from 1 to24 PEG molecules on one NTSC-PYY peptide. Said conjugates may furtherinclude suitable linkers between the PEG molecules and the NTSC-PYYmolecule, including, but not limited to γ-glutamate, —NHC(O), C(O), andC₍₁₋₄₎alkyl.

As used herein, the phrase “lipidation” refers to covalent conjugates ofan NTSC-PYY peptide and one or more lipophilic groups. Preferredlipophilic groups include long chain hydrocarbon groups. Otherlipophilic groups include steroids, terpenes, fat soluble vitamins,phytosterols, terpenoids, phospholipids, glycerols, and natural orsynthetic fatty acids. Examples of lipophilic groups include, but arenot limited to α-tocopherolyl, stearic acid, palmitic acid, andarachidic acid. Said conjugates may further include suitable linkersbetween the lipophilic molecules and the NTSC-PYY molecule, including,but not limited to γ-glutamate, —NHC(O), C(O), and C(1.4)alkyl.

The term “PYY₃₋₃₆” shall refer to the following compound (SEQ ID NO:111):

Pharmaceutical Compositions

In another general aspect, the invention relates to a pharmaceuticalcomposition, comprising the conjugates and compounds of the inventionand a pharmaceutically acceptable carrier. The term “pharmaceuticalcomposition” as used herein means a product comprising a conjugate ofthe invention together with a pharmaceutically acceptable carrier.Conjugates and compounds of the invention and compositions comprisingthem are also useful in the manufacture of a medicament for therapeuticapplications mentioned herein.

As used herein, the term “carrier” refers to any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipidcontaining vesicle, microsphere, liposomal encapsulation, or othermaterial well known in the art for use in pharmaceutical formulations.It will be understood that the characteristics of the carrier, excipientor diluent will depend on the route of administration for a particularapplication. As used herein, the term “pharmaceutically acceptablecarrier” refers to a non-toxic material that does not interfere with theeffectiveness of a composition according to the invention or thebiological activity of a composition according to the invention.According to particular embodiments, in view of the present disclosure,any pharmaceutically acceptable carrier suitable for use in an antibodypharmaceutical composition can be used in the invention.

Pharmaceutically acceptable acidic/anionic salts for use in theinvention include, and are not limited to acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate,carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate, mesylate, methylbromide,methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate,tosylate and triethiodide. Organic or inorganic acids also include, andare not limited to, hydriodic, perchloric, sulfuric, phosphoric,propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,saccharinic or trifluoroacetic acid.

Pharmaceutically acceptable basic/cationic salts include, and are notlimited to aluminum, 2-amino-2-hydroxymethyl -propane-1,3-diol (alsoknown as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”),ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline,cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine,magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium,procaine, quinine, sodium, triethanolamine, or zinc.

In some embodiments of the invention, pharmaceutical formulations areprovided comprising the compounds of the invention in an amount fromabout 0.001 mg/ml to about 100 mg/ml, from about 0.01 mg/ml to about 50mg/ml, or from about 0.1 mg/ml to about 25 mg/ml. The pharmaceuticalformulation may have a pH from about 3.0 to about 10, for example fromabout 3 to about 7, or from about 5 to about 9. The formulation mayfurther comprise at least one ingredient selected from the groupconsisting of a buffer system, preservative(s), tonicity agent(s),chelating agent(s), stabilizer(s) and surfactant(s).

The formulation of pharmaceutically active ingredients withpharmaceutically acceptable carriers is known in the art, e.g.,Remington: The Science and Practice of Pharmacy (e.g. 21st edition(2005), and any later editions). Non-limiting examples of additionalingredients include: buffers, diluents, solvents, tonicity regulatingagents, preservatives, stabilizers, and chelating agents. One or morepharmaceutically acceptable carrier may be used in formulating thepharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is aliquid formulation. A preferred example of a liquid formulation is anaqueous formulation, i.e., a formulation comprising water. The liquidformulation may comprise a solution, a suspension, an emulsion, amicroemulsion, a gel, and the like. An aqueous formulation typicallycomprises at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%,90%, or at least 95% w/w of water.

In one embodiment, the pharmaceutical composition may be formulated asan injectable which can be injected, for example, via an injectiondevice (e.g., a syringe or an infusion pump). The injection may bedelivered subcutaneously, intramuscularly, intraperitoneally, orintravenously, for example.

In another embodiment, the pharmaceutical composition is a solidformulation, e.g., a freeze-dried or spray-dried composition, which maybe used as is, or whereto the physician or the patient adds solvents,and/or diluents prior to use. Solid dosage forms may include tablets,such as compressed tablets, and/or coated tablets, and capsules (e.g.,hard or soft gelatin capsules). The pharmaceutical composition may alsobe in the form of sachets, dragees, powders, granules, lozenges, orpowders for reconstitution, for example.

The dosage forms may be immediate release, in which case they maycomprise a water-soluble or dispersible carrier, or they may be delayedrelease, sustained release, or modified release, in which case they maycomprise water-insoluble polymers that regulate the rate of dissolutionof the dosage form in the gastrointestinal tract.

In other embodiments, the pharmaceutical composition may be deliveredintranasally, intrabuccally, or sublingually.

The pH in an aqueous formulation can be between pH 3 and pH 10. In oneembodiment of the invention, the pH of the formulation is from about 7.0to about 9.5. In another embodiment of the invention, the pH of theformulation is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical compositioncomprises a buffer. Non-limiting examples of buffers include: arginine,aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaricacid, glycine, glycylglycine, histidine, lysine, maleic acid, malicacid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate,sodium phosphate, succinate, tartaric acid, tricine, andtris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer maybe present individually or in the aggregate, in a concentration fromabout 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml toabout 20 mg/ml. Pharmaceutical compositions comprising each one of thesespecific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a preservative. Non-limiting examples of buffers include:benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine,chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate,imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol,2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate,thiomerosal, and mixtures thereof. The preservative may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificpreservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises an isotonic agent. Non-limiting examples of the embodimentinclude a salt (such as sodium chloride), an amino acid (such asglycine, histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, and threonine), an alditol (such as glycerol,1,2-propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol),polyethyleneglycol (e.g. PEG400), and mixtures thereof. Another exampleof an isotonic agent includes a sugar. Non-limiting examples of sugarsmay be mono-, di-, or polysaccharides, or water-soluble glucans,including for example fructose, glucose, mannose, sorbose, xylose,maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch,and sodium carboxymethylcellulose. Another example of an isotonic agentis a sugar alcohol, wherein the term “sugar alcohol” is defined as aC(4-8) hydrocarbon having at least one —OH group. Non-limiting examplesof sugar alcohols include mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. Pharmaceutical compositions comprisingeach isotonic agent listed in this paragraph constitute alternativeembodiments of the invention. The isotonic agent may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificisotonic agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a chelating agent. Non-limiting examples of chelating agentsinclude citric acid, aspartic acid, salts of ethylenediaminetetraaceticacid (EDTA), and mixtures thereof. The chelating agent may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificchelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer. Non-limiting examples of stabilizers include oneor more aggregation inhibitors, one or more oxidation inhibitors, one ormore surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer, wherein said stabilizer iscarboxy-/hydroxycellulose and derivates thereof (such as HPC, HPC-SL,HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol(such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone,salts (such as sodium chloride), sulphur-containing substances such asmonothioglycerol), or thioglycolic acid. The stabilizer may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificstabilizers constitute alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical compositioncomprises one or more surfactants, preferably a surfactant, at least onesurfactant, or two different surfactants. The term “surfactant” refersto any molecules or ions that are comprised of a water-soluble(hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactantmay, for example, be selected from the group consisting of anionicsurfactants, cationic surfactants, nonionic surfactants, and/orzwitterionic surfactants. The surfactant may be present individually orin the aggregate, in a concentration from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificsurfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical compositioncomprises one or more protease inhibitors, such as, e.g., EDTA, and/orbenzamidine hydrochloric acid (HCl). The protease inhibitor may bepresent individually or in the aggregate, in a concentration from about0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising eachone of these specific protease inhibitors constitute alternativeembodiments of the invention.

The pharmaceutical composition of the invention may comprise an amountof an amino acid base sufficient to decrease aggregate formation of thepolypeptide during storage of the composition. The term “amino acidbase” refers to one or more amino acids (such as methionine, histidine,imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine), or analogues thereof. Any amino acid may be present eitherin its free base form or in its salt form. Any stereoisomer (i.e., L, D,or a mixture thereof) of the amino acid base may be present. The aminoacid base may be present individually or in the combination with otheramino acid bases, in a concentration from about 0.01 mg/ml to about 50mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.Pharmaceutical compositions comprising each one of these specific aminoacid bases constitute alternative embodiments of the invention.

It is also apparent to one skilled in the art that the therapeuticallyeffective dose for compounds of the present invention or apharmaceutical composition thereof will vary according to the desiredeffect. Therefore, optimal dosages to be administered may be readilydetermined by one skilled in the art and will vary with the particularcompound used, the mode of administration, the strength of thepreparation, and the advancement of the disease condition. In addition,factors associated with the particular subject being treated, includingsubject age, weight, diet and time of administration, will result in theneed to adjust the dose to an appropriate therapeutic level.

For all indications, the compounds of the invention are preferablyadministered peripherally at a dose of about 1 μg to about 5 mg per dayin single or divided doses (e.g., a single dose can be divided into 2,3, 4, 5, 6, 7, 8, 9, or 10 subdoses), or at about 0.01 μg/kg to about500 μg/kg per dose, more preferably about 0.05 μg/kg to about 250 μg/kg,most preferably below about 50 μg/kg. Dosages in these ranges will varywith the potency of each agonist, of course, and are readily determinedby one of skill in the art. The above dosages are thus exemplary of theaverage case. There can, of course, be individual instances where higheror lower dosage ranges are merited, and such are within the scope ofthis invention.

In certain embodiments, the compounds of the invention are administeredat a dose of about 1 μg to about 5 mg, or at a dose of about 0.01 μg/kgto about 500 μg/kg, more preferably at a dose of about 0.05 μg/kg toabout 250 μg/kg, most preferably at a dose below about 50 μg/kg with adose of a second therapeutic agent (e.g., liraglutide) at a dose ofabout 1 μg to about 5 mg, or at a dose of about 0.01 μg/kg to about 500μg/kg, more preferably at a dose of about 0.05 μg/kg to about 250 μg/kg,most preferably at a dose below about 50 μg/kg.

The pharmaceutically-acceptable salts of the compounds of the inventioninclude the conventional non-toxic salts or the quaternary ammoniumsalts which are formed from inorganic or organic acids or bases.Examples of such acid addition salts include acetate, adipate, benzoate,benzenesulfonate, citrate, camphorate, dodecylsulfate, hydrochloride,hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate,pivalate, propionate, succinate, sulfate and tartrate. Base saltsinclude ammonium salts, alkali metal salts such as sodium and potassiumsalts, alkaline earth metal salts such as calcium and magnesium salts,salts with organic bases such as dicyclohexylamino salts and salts withamino acids such as arginine. Also, the basic nitrogen-containing groupsmay be quaternized with, for example, alkyl halides.

The pharmaceutical compositions of the invention may be administered byany means that accomplish their intended purpose. Examples includeadministration by parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, buccal or ocular routes. Administrationmay be by the oral route. Suitable formulations for parenteraladministration include aqueous solutions of the active conjugates inwater-soluble form, for example, water-soluble salts, acidic solutions,alkaline solutions, dextrose-water solutions, isotonic carbohydratesolutions and cyclodextrin inclusion complexes.

The present invention also encompasses a method of making apharmaceutical composition comprising mixing a pharmaceuticallyacceptable carrier with any of the compounds of the present invention.Additionally, the present invention includes pharmaceutical compositionsmade by mixing one or more pharmaceutically acceptable carriers with anyof the compounds of the present invention.

Furthermore, the compounds of the present invention may have one or morepolymorph or amorphous crystalline forms and as such are intended to beincluded in the scope of the invention. In addition, the compounds mayform solvates, for example with water (i.e., hydrates) or common organicsolvents. As used herein, the term “solvate” means a physicalassociation of the compounds of the present invention with one or moresolvent molecules. This physical association involves varying degrees ofionic and covalent bonding, including hydrogen bonding. In certaininstances the solvate will be capable of isolation, for example when oneor more solvent molecules are incorporated in the crystal lattice of thecrystalline solid. The term “solvate” is intended to encompass bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.

It is intended that the present invention include within its scopepolymorphs and solvates of the conjugates of the present invention.Thus, in the methods of treatment of the present invention, the term“administering” shall encompass the means for treating, ameliorating orpreventing a syndrome, disorder or disease described herein with theconjugates of the present invention or a polymorph or solvate thereof,which would obviously be included within the scope of the inventionalbeit not specifically disclosed.

In another embodiment, the invention relates to the compounds of theinvention for use as a medicament.

The present invention includes within its scope prodrugs of thecompounds of this invention. In general, such prodrugs will befunctional derivatives of the compounds which are readily convertible invivo into the required compound. Thus, in the methods of treatment ofthe present invention, the term “administering” shall encompass thetreatment of the various disorders described with the compoundspecifically disclosed or with a compound which may not be specificallydisclosed, but which converts to the specified compound in vivo afteradministration to the patient. Conventional procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, Ed. H. Bundgaard, Elsevier, 1985.

Furthermore, it is intended that within the scope of the presentinvention, any element, in particular when mentioned in relation to thecompounds of the invention, shall comprise all isotopes and isotopicmixtures of said element, either naturally occurring or syntheticallyproduced, either with natural abundance or in an isotopically enrichedform. For example, a reference to hydrogen includes within its scope ¹H,²H (D), and ³H (T). Similarly, references to carbon and oxygen includewithin their scope respectively ¹²C, ¹³C and ¹⁴C and ¹⁶O and ¹⁸O. Theisotopes may be radioactive or non-radioactive. Radiolabeled compoundsof the invention may comprise a radioactive isotope selected from thegroup of ³H, ¹¹C, ¹⁸F, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and⁸²Br. Preferably, the radioactive isotope is selected from the group of³H, ¹¹C and ¹⁸F.

Some compounds of the present invention may exist as atropisomers.Atropisomers are stereoisomers resulting from hindered rotation aboutsingle bonds where the steric strain barrier to rotation is high enoughto allow for the isolation of the conformers. It is to be understoodthat all such conformers and mixtures thereof are encompassed within thescope of the present invention.

Where the compounds according to this invention have at least one stereocenter, they may accordingly exist as enantiomers or diastereomers. Itis to be understood that all such isomers and mixtures thereof areencompassed within the scope of the present invention.

Where the processes for the preparation of the compounds according tothe invention give rise to mixture of stereoisomers, these isomers maybe separated by conventional techniques such as preparativechromatography. The compounds may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their component enantiomers by standard techniques, such as theformation of diastereomeric pairs by salt formation with an opticallyactive acid, such as (−)-di-p-toluoyl-D-tartaric acid and/or(+)-di-p-toluoyl-L-tartaric acid followed by fractional crystallizationand regeneration of the free base. The compounds may also be resolved byformation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral column viahigh performance liquid chromatography (HPLC) or SFC. In some instancesrotamers of compounds may exist which are observable by 1H NMR leadingto complex multiplets and peak integration in the 1H NMR spectrum.

During any of the processes for preparation of the compounds of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie,Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups inOrganic Synthesis, John Wiley & Sons, 1991, each of which is hereinincorporated by reference in its entirety for all purposes. Theprotecting groups may be removed at a convenient subsequent stage usingmethods known from the art.

Methods of Use

The present invention is directed to a method for preventing, treatingor ameliorating a Y2 receptor mediated syndrome, disorder or disease ina subject in need thereof comprising administering to the subject inneed thereof an effective amount of a compound, a derivative or apharmaceutically acceptable salt thereof, optionally conjugated to ahalf-life extension moiety, or a pharmaceutical composition of theinvention.

The present invention also provides a method for preventing, treating,delaying the onset of, or ameliorating a disorder, disease, or conditionor any one or more symptoms of said disorder, disease, or condition in asubject in need thereof, comprising administering to the subject in needthereof an effective amount of a compound, a derivative or apharmaceutically acceptable salt thereof, optionally conjugated to ahalf-life extension moiety, or a pharmaceutical composition of theinvention.

According to particular embodiments, the disease disorder, or conditionis selected from the group consisting of obesity, type I or II diabetes,metabolic syndrome (i.e., Syndrome X), insulin resistance, impairedglucose tolerance (e.g., glucose intolerance), hyperglycemia,hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenitalhyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabeticnephropathy, and other cardiovascular risk factors such as hypertensionand cardiovascular risk factors related to unmanaged cholesterol and/orlipid levels, osteoporosis, inflammation, non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease,and/or eczema.

According to particular embodiments, a therapeutically effective amountrefers to the amount of therapy which is sufficient to achieve one, two,three, four, or more of the following effects: (i) reduce or amelioratethe severity of the disease, disorder, or condition to be treated or asymptom associated therewith; (ii) reduce the duration of the disease,disorder or condition to be treated, or a symptom associated therewith;(iii) prevent the progression of the disease, disorder or condition tobe treated, or a symptom associated therewith; (iv) cause regression ofthe disease, disorder or condition to be treated, or a symptomassociated therewith; (v) prevent the development or onset of thedisease, disorder or condition to be treated, or a symptom associatedtherewith; (vi) prevent the recurrence of the disease, disorder orcondition to be treated, or a symptom associated therewith; (vii) reducehospitalization of a subject having the disease, disorder or conditionto be treated, or a symptom associated therewith; (viii) reducehospitalization length of a subject having the disease, disorder orcondition to be treated, or a symptom associated therewith; (ix)increase the survival of a subject with the disease, disorder orcondition to be treated, or a symptom associated therewith; (xi) inhibitor reduce the disease, disorder or condition to be treated, or a symptomassociated therewith in a subject; and/or (xii) enhance or improve theprophylactic or therapeutic effect(s) of another therapy.

The therapeutically effective amount or dosage can vary according tovarious factors, such as the disease, disorder or condition to betreated, the means of administration, the target site, the physiologicalstate of the subject (including, e.g., age, body weight, health),whether the subject is a human or an animal, other medicationsadministered, and whether the treatment is prophylactic or therapeutic.Treatment dosages are optimally titrated to optimize safety andefficacy.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related the disease, disorder, orcondition, which is not necessarily discernible in the subject, but canbe discernible in the subject. The terms “treat,” “treating,” and“treatment,” can also refer to causing regression, preventing theprogression, or at least slowing down the progression of the disease,disorder, or condition. In a particular embodiment, “treat,” “treating,”and “treatment” refer to an alleviation, prevention of the developmentor onset, or reduction in the duration of one or more symptomsassociated with the disease, disorder, or condition. In a particularembodiment, “treat,” “treating,” and “treatment” refer to prevention ofthe recurrence of the disease, disorder, or condition. In a particularembodiment, “treat,” “treating,” and “treatment” refer to an increase inthe survival of a subject having the disease, disorder, or condition. Ina particular embodiment, “treat,” “treating,” and “treatment” refer toelimination of the disease, disorder, or condition in the subject.

In one embodiment, the invention provides a method for preventing,treating, delaying the onset of, or ameliorating obesity, or any one ormore symptoms of obesity in a subject in need thereof, the methodcomprising administering to the subject in need thereof an effectiveamount of a compound, a derivative or a pharmaceutically acceptable saltthereof, optionally conjugated to a half-life extension moiety, or apharmaceutical composition of the invention. In some embodiments, thebody weight of a subject is reduced, for example, by between about 0.01%to about 0.1%, between about 0.1% to about 0.5%, between about 0.5% toabout 1%, between about 1% to about 5%, between about 2% to about 3%,between about 5% to about 10%, between about 10% to about 15%, betweenabout 15% to about 20%, between about 20% to about 25%, between about25% to about 30%, between about 30% to about 35%, between about 35% toabout 40%, between about 40% to about 45%, or between about 45% to about50%, relative to the body weight of a subject prior to administration ofany of the conjugates, compounds, pharmaceutical compositions, forms, ormedicaments of the invention described herein, or compared to controlsubjects not receiving any of the conjugates, compositions, forms,medicaments, or combinations of the invention described herein.

In some embodiments, the reduction in body weight is maintained forabout 1 week, for about 2 weeks, for about 3 weeks, for about 1 month,for about 2 months, for about 3 months, for about 4 months, for about 5months, for about 6 months, for about 7 months, for about 8 months, forabout 9 months, for about 10 months, for about 11 months, for about 1year, for about 1.5 years, for about 2 years, for about 2.5 years, forabout 3 years, for about 3.5 years, for about 4 years, for about 4.5years, for about 5 years, for about 6 years, for about 7 years, forabout 8 years, for about 9 years, for about 10 years, for about 15years, or for about 20 years, for example.

The present invention provides a method of preventing, treating,delaying the onset of, or ameliorating a syndrome, disorder or disease,or any one or more symptoms of said syndrome, disorder, or disease in asubject in need thereof, wherein said syndrome, disorder or disease isselected from the group consisting of obesity, type I or type IIdiabetes, metabolic syndrome (i.e., Syndrome X), insulin resistance,impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia,hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenitalhyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabeticnephropathy, and other cardiovascular risk factors such as hypertensionand cardiovascular risk factors related to unmanaged cholesterol and/orlipid levels, osteoporosis, inflammation, non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease,and eczema, comprising administering to the subject in need thereof aneffective amount of a compound, a derivative or a pharmaceuticallyacceptable salt thereof, optionally conjugated to a half-life extensionmoiety, or a pharmaceutical composition of the invention.

As used herein, metabolic syndrome refers to a subject having any one ormore of the following: high blood sugar (e.g., high fasting bloodsugar), high blood pressure, abnormal cholesterol levels (e.g., low HDLlevels), abnormal triglyceride levels (e.g., high triglycerides), alarge waistline (i.e., waist circumference), increased fat in theabdominal area, insulin resistance, glucose intolerance, elevatedC-reactive protein levels (i.e., a proinflammatory state), and increasedplasma plasminogen activator inhibitor-1 and fibrinogen levels (i.e., aprothrombotic state).

The present invention provides a method of reducing food intake in asubject in need thereof, the method comprising administering to thesubject in need thereof an effective amount of a compound, a derivativeor a pharmaceutically acceptable salt thereof, optionally conjugated toa half-life extension moiety, or a pharmaceutical composition of theinvention. In some embodiments, food intake of a subject is reduced, forexample, by between about 0.01% to about 0.1%, between about 0.1% toabout 0.5%, between about 0.5% to about 1%, between about 1% to about5%, between about 2% to about 3%, between about 5% to about 10%, betweenabout 10% to about 15%, between about 15% to about 20%, between about20% to about 25%, between about 25% to about 30%, between about 30% toabout 35%, between about 35% to about 40%, between about 40% to about45%, or between about 45% to about 50%, relative to food intake of asubject prior to administration of any of the conjugates, compounds,compositions, forms, medicaments, or combinations of the inventiondescribed herein, or compared to control subjects not receiving any ofthe conjugates, compounds, compositions, forms, medicaments, orcombinations of the invention described herein.

In some embodiments, the reduction in food intake is maintained forabout 1 week, for about 2 weeks, for about 3 weeks, for about 1 month,for about 2 months, for about 3 months, for about 4 months, for about 5months, for about 6 months, for about 7 months, for about 8 months, forabout 9 months, for about 10 months, for about 11 months, for about 1year, for about 1.5 years, for about 2 years, for about 2.5 years, forabout 3 years, for about 3.5 years, for about 4 years, for about 4.5years, for about 5 years, for about 6 years, for about 7 years, forabout 8 years, for about 9 years, for about 10 years, for about 15years, or for about 20 years, for example.

The present invention provides a method of reducing glycated hemoglobin(A1C) in a subject in need thereof, the method comprising administeringto the subject in need thereof an effective amount of a compound, aderivative or a pharmaceutically acceptable salt thereof, optionallyconjugated to a half-life extension moiety, or a pharmaceuticalcomposition of the invention. In some embodiments, A1C of a subject isreduced, for example, by between about 0.001% and about 0.01%, betweenabout 0.01% and about 0.1%, between about 0.1% and about 0.2%, betweenabout 0.2% and about 0.3%, between about 0.3% and about 0.4%, betweenabout 0.4% and about 0.5%, between about 0.5% and about 1%, betweenabout 1% and about 1.5%, between about 1.5% and about 2%, between about2% and about 2.5%, between about 2.5% and about 3%, between about 3% andabout 4%, between about 4% and about 5%, between about 5% and about 6%,between about 6% and about 7%, between about 7% and about 8%, betweenabout 8% and about 9%, or between about 9% and about 10% relative to theA1C of a subject prior to administration of any of the conjugates,compounds, compositions, forms, medicaments, or combinations of theinvention described herein, or compared to control subjects notreceiving any of the conjugates, compounds, compositions, forms,medicaments, or combinations of the invention described herein.

In other embodiments, methods are provided for reducing fasting bloodglucose levels in a subject in need thereof, the methods comprisingadministering to the subject in need thereof an effective amount of acompound, a derivative or a pharmaceutically acceptable salt thereof,optionally conjugated to a half-life extension moiety, or apharmaceutical composition of the invention. Fasting blood glucoselevels may be reduced to less than about 140 to about 150 mg/dL, lessthan about 140 to about 130 mg/dL, less than about 130 to about 120mg/dL, less than about 120 to about 110 mg/dL, less than about 110 toabout 100 mg/dL, less than about 100 to about 90 mg/dL, or less thanabout 90 to about 80 mg/dL, relative to the fasting blood glucose levelsof a subject prior to administration of any of the conjugates,compounds, compositions, forms, medicaments, or combinations of theinvention described herein, or compared to control subjects notreceiving any of the conjugates, compounds, compositions, forms,medicaments, or combinations of the invention described herein.

The present invention provides a method of modulating Y2 receptoractivity in a subject in need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acompound, a derivative or a pharmaceutically acceptable salt thereof,optionally conjugated to a half-life extension moiety, or apharmaceutical composition of the invention. As used herein,“modulating” refers to increasing or decreasing receptor activity.

In some embodiments, an effective amount of a compound, a derivative ora pharmaceutically acceptable salt thereof, optionally conjugated to ahalf-life extension moiety, or a pharmaceutical composition of theinvention is administered to a subject in need thereof once daily, twicedaily, three times daily, four times daily, five times daily, six timesdaily, seven times daily, or eight times daily. In other embodiments, aneffective amount of a compound, a derivative or a pharmaceuticallyacceptable salt thereof, optionally conjugated to a half-life extensionmoiety, or a pharmaceutical composition of the invention is administeredto a subject in need thereof once every other day, once per week, twiceper week, three times per week, four times per week, five times perweek, six times per week, two times per month, three times per month, orfour times per month.

Another embodiment of the invention comprises a method of preventing,treating, delaying the onset of, or ameliorating a disease, disorder orsyndrome, or one or more symptoms of any of said diseases, disorders, orsyndromes in a subject in need thereof, the method comprisingadministering to the subject in need thereof an effective amount of acompound, a derivative or a pharmaceutically acceptable salt thereof,optionally conjugated to a half-life extension moiety, or apharmaceutical composition of the invention in a combination therapy. Incertain embodiments, the combination therapy is a second therapeuticagent. In certain embodiments, the combination therapy is a surgicaltherapy.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy.

As used herein, combination therapy refers to administering to a subjectin need thereof one or more additional therapeutic agents, or one ormore surgical therapies, concurrently with an effective amount of acompound, a derivative or a pharmaceutically acceptable salt thereof,optionally conjugated to a half-life extension moiety, or apharmaceutical composition of the invention. In some embodiments, theone or more additional therapeutic agents or surgical therapies can beadministered on the same day as an effective amount of a compound, aderivative or a pharmaceutically acceptable salt thereof, optionallyconjugated to a half-life extension moiety, or a pharmaceuticalcomposition of the invention, and in other embodiments, the one or moreadditional therapeutic agents or surgical therapies may be administeredin the same week or the same month as an effective amount of a compound,a derivative or a pharmaceutically acceptable salt thereof, optionallyconjugated to a half-life extension moiety, or a pharmaceuticalcomposition of the invention.

In certain embodiments, wherein the disease or disorder is selected fromthe group consisting of obesity, type II diabetes, metabolic syndrome,insulin resistance and dyslipidemia, the second therapeutic agent can bean antidiabetic agent. In certain embodiments, the antidiabetic agentcan be a glucagon-like peptide-1 (GLP-1) receptor modulator.

The present invention also contemplates preventing, treating, delayingthe onset of, or ameliorating any of the diseases, disorders, syndromes,or symptoms described herein in a subject in need thereof with acombination therapy that comprises administering to the subject in needthereof an effective amount of a compound, a derivative or apharmaceutically acceptable salt thereof, optionally conjugated to ahalf-life extension moiety, or a pharmaceutical composition of theinvention, in combination with any one or more of the followingtherapeutic agents: a dipeptidyl peptidase-4 (DPP-4) inhibitor (e.g.,sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); a GLP-1receptor agonist (e.g., short-acting GLP-1 receptor agonists such asexenatide and lixisenatide; intermediate-acting GLP-1 receptor agonistssuch as liraglutide; long-acting GLP-1 receptor agonists such asexenatide extended-release, albiglutide, dulaglutide); a sodium-glucoseco-transporter-2 (SGLT-2) inhibitors (e.g., canaglifozin, dapaglifozin,empaglifozin, etc.); bile acid sequestrants (e.g., colesevelam, etc.);dopamine receptor agonists (e.g., bromocriptine quick-release);biguanides (e.g., metformin, etc.); insulin; oxyntomodulin;sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide,glibenclamide, glibornuride, glisoxepide, glyclopyramide, tolazamide,tolbutamide, acetohexamide, carbutamide, etc.); and thiazolidinediones(e.g; pioglitazone, rosiglitazone, lobeglitazone, ciglitazone,darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone,etc.). In some embodiments, the dose of the additional therapeuticagent(s) is reduced when given in combination with a compound, aderivative or a pharmaceutically acceptable salt thereof, optionallyconjugated to a half-life extension moiety, or a pharmaceuticalcomposition of the invention. In some embodiments, when used incombination with a conjugate or compound of the invention, theadditional therapeutic agent(s) may be used in lower doses than wheneach is used singly.

In certain embodiments, wherein the disease or disorder is selected fromthe group consisting of obesity, type I or type II diabetes, metabolicsyndrome (i.e., Syndrome X), insulin resistance, impaired glucosetolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia,hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism(CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and othercardiovascular risk factors such as hypertension and cardiovascular riskfactors related to unmanaged cholesterol and/or lipid levels,osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), renal disease, and eczema, thesecond therapeutic agent can be liraglutide.

The present invention contemplates preventing, treating, delaying theonset of, or ameliorating any of the diseases, disorders, syndromes, orsymptoms described herein in a subject in need thereof, with acombination therapy that comprises administering to the subject in needthereof an effective amount of a compound, a derivative or apharmaceutically acceptable salt thereof, optionally conjugated to ahalf-life extension moiety, or a pharmaceutical composition of theinvention in combination with a surgical therapy. In certainembodiments, the surgical therapy can be bariatric surgery (e.g.,gastric bypass surgery, such as Roux-en-Y gastric bypass surgery; sleevegastrectomy; adjustable gastric band surgery; biliopancreatic diversionwith duodenal switch; intragastric balloon; gastric plication; andcombinations thereof).

In embodiments in which the one or more additional therapeutic agents orsurgical therapies is administered on the same day as an effectiveamount of a conjugate or compound of the invention, the conjugate orcompound of the invention may be administered prior to, after, orsimultaneously with the additional therapeutic agent or surgicaltherapy. The use of the term “in combination” does not restrict theorder in which therapies are administered to a subject. For example, afirst therapy (e.g., a composition described herein) can be administeredprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g.,5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksafter) the administration of a second therapy to a subject.

Abbreviations

Herein and throughout the application, the following abbreviations maybe used. Abu: 4-aminobutyric acid; Ac₂O: acetic anhydride; aq: aqueous;alloc: allyloxycarbonyl; arach: arachidoyl; Boc: tert-butoxycarbonyl;BSA: bovine serum albumin; CDI: 1,1′-carbonyldiimidazole; DCM:dichloromethane; Dde: 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)ethyl;DIBAL-H: diisobutylaluminum hydride; DIC: diisopropylcarbodiimide; DIEA:diisopropylethylamine; DMA: N, N-dimethylacetamide; DMF: N,N-dimethylformamide; DMSO: methyl sulfoxide; DODT:2,2′-(ethylenedioxy)diethanethiol; EDC:N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; EDCI:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Et: ethyl;EtOAc: ethyl acetate; EtOH: ethyl alcohol; FBS: fetal bovine serum;Fmoc: 9-fluorenylmethyloxycarbonyl; g: gram(s); h: hour(s); HATU:2-(1H-7-azabenztriazol-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate; HBSS: Hank's balanced salt solution; HBTU:2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate); HCTU:2-(6-chloro-1H-benztriazol-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate; HCl: hydrochloric acid; HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOBT:1-hydroxybenztriazole; HPLC: high performance liquid chromatography;ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)-3-methylbutyl; LAH:lithium aluminum hydride; LCMS: high pressure liquid chromatography withmass spectrometer; Me: methyl; MeCN: acetonitrile; MeOH: methyl alcohol;mn: milligram; min: minute(s); Mmt: 4-methoxytrityl; mpm: mL per minute;Mtt: 4-methyltrityl; NHS: N-hydroxysuccinimide; NMP:1-methyl-2-pyrrolidone; OEG: 8-amino-3,6-dioxaoctanoyl; Oxyma: ethylcyano(hydroxyimino)acetate; Pal: palmitoyl; Pbf:2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Pd(PPh₃)₄:tetrakis(triphenylphosphine)palladium(0); PhSiH₃: phenylsilane; psi:reduced amide bond (between adjacent amino acids); PyBroP:bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; Pyoxim:1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphoniumhexafluorophosphate; rt: room temperature; RT: retention time; satd.:saturated; SPPS: solid phase peptide synthesis; Stear: stearoyl; t-Bu:tert-butyl; TBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate; TFA: trifluoroacetic acid; THF: tetrahydrofuran; THP:tetrahydropyranyl; TIPS: triisopropylsilane; Tris:tris(hydroxymethyl)aminomethane; Trt: triphenylmethyl

Synthesis

Compounds of Formula I in the present invention can be synthesized inaccordance with the general synthetic methods known to those who areskilled in the art. The following description of the synthesis is forexemplary purposes and is in no way meant to be a limit of theinvention.

The NTSC cyclic PYY (NTSC-PYY) analogues or derivatives of thisinvention may be synthesized by a variety of known, conventionalprocedures for the formation of successive peptide linkages betweenamino acids, and are preferentially carried out by solid phase peptidesynthesis (SPPS), as generally described by Merrifield (J. Am. Chem.Soc., 1963, 85, 2149-2154), using an automated peptide synthesizer,traditional bench synthesis, or a combination of both approaches.Conventional procedures for peptide synthesis involve the condensationbetween the free amino group of one amino acid residue, whose otherreactive functionalities have been suitably protected, and the freecarboxyl group of another amino acid, whose reactive functionalitieshave also been suitably protected. Examples of condensation agentstypically utilized for peptide bond formation includediisopropylcarbodiimide (DIC) with or without 1-hydroxybenztriazole(HOBT) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU), 2-(1H-7-azabenztriazol-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HATU),2-(6-chloro-1H-benztriazol-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),1-Cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyOxim),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate(TBTU) bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),and the like.

The automated peptide synthetic methodology may be carried out at roomtemperature, or at elevated temperatures, preferably through theapplication of microwave heating, as described by Yu (J. Org. Chem.,1992, 57, 4781-4784) and as more recently refined by Palasek (J. Pept.Sci., 2007, 13, 143-148).

Compounds of the present invention (C-terminal amides) can beconveniently prepared using N-α-Fmoc protected amino acid methodology,whereby the carboxy terminus of a suitably protected N-α-Fmoc protectedamino acid is coupled onto a conventional solid phase resin using asuitable coupling agent. Suitable conventional, commercially-availablesolid phase resins include Rink amide MBHA resin, Rink amide AM resin,Tentagel S RAM Resin, Fmoc-PAL-PEG PS resin, SpheriTide Rink amideresin, ChemMatrix Rink resin, Sieber amide resin, TG Sieber resin andthe like. The resin-bound Fmoc-amino acid may then be deprotected byexposure to 20% piperidine in either DMF or NMP, treatment of whichserves to selectively remove the Fmoc protecting group. AdditionalFmoc-protected amino acids are then subsequently coupled and deprotectedsequentially, thereby generating the desired resin-bound protectedpeptide. In certain instances, it may be necessary to utilize anorthogonally reactive protecting group for another amine in the peptidesequence that would withstand the Fmoc deprotection conditions.Protecting groups such 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt),both removable by 1% TFA/DCM treatments, or preferably allyloxycarbonyl(alloc; removable by Pd(PPh₃)₄/PhSiH₃ treatment),1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)ethyl (Dde; removable bytreatment with 2-3% hydrazine/DMF) and1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)-3-methylbutyl (ivDde;removable by treatment with 2-3% hydrazine/DMF) can be used effectivelyin such instances.

In conventional peptide synthetic methodologies, reactive side chains ofalpha amino acids are generally protected throughout the synthesis withsuitable protecting groups to render them inert to the coupling anddeprotection protocols. While multiple protecting groups for amino acidside chains are known in the art, herein the following protecting groupsare most preferred: tert-butyl (t-Bu) for serine, threonine, glutamicacid, aspartic acid and tyrosine; trityl (Trt) for asparagine,glutamine, cysteine, homocysteine and histidine; tert-butyloxycarbonyl(Boc) for tryptophan and the ε-amino group of lysine; and2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine.These protecting groups are removed upon strong acid treatment, such aswith high concentrations of trifluoroacetic acid (TFA).

Upon completion of the SPPS, the resin-bound, side chain-protectedpeptide is deprotected and concomitantly cleaved from the resin using acleavage cocktail that consists predominantly of (TFA) along withvarious combinations of carbocation scavengers, such astriisopropylsilane (TIPS), water, phenol and anisole. The crude solidpeptide is then isolated by precipitation of the peptide/cocktailfiltrate with cold ether. In the special case of Sieber resin-boundprotected peptides, cleavage of the protected peptide from the resin maybe advantageously effected upon repeated treatment with 1-2% TFA in DCMwithout causing side chain deprotections. Once isolated, furthermanipulations of the protected peptide may be carried out in solutionphase reactions. Finally, the protected peptide may be globallydeprotected using a separate treatment with the cleavage cocktail andprecipitated as described above. The crude peptide thus obtained is thendissolved at low concentration (ca., <4 mg/mL) in a largely aqueoussolvent system containing an organic co-solvent such as acetonitrile orethanol. Upon raising the pH of the solution to a >5, the peptide thenundergoes an intramolecular cyclization reaction to form thecorresponding crude NTSC PYY analogue of the present invention. NTSC PYYanalogues thus formed may be purified using purification techniquesgenerally known in the art. A preferable method of peptide purificationused herein is reverse phase high pressure liquid chromatography (HPLC).Purified peptides are then characterized by liquid chromatography/massspectrometry (LC/MS).

General Schemes

A general synthetic procedure for the synthesis of C-terminal amidesNTSC-PYY peptides wherein the BRIDGE is -Ph-CH₂—S— is shown in Scheme 1.

A) Synthesis of Resin-Bound C-Terminal Amide Peptide

The protected peptidyl resin can be synthesized using Fmoc strategy asdescribed above on a CEM Liberty Blue Microwave peptide synthesizerusing low loading Rink amide resins, preferably, Fmoc-PAL-PEG PS resin(ca., 0.16-0.2 meq/g, supplied by Applied Biosystems) on a scale of 0.1mmol, as depicted in Scheme 1. Standard Fmoc-protected amino acids(supplied by Novabiochem (EMD Millipore), Bachem, Peptides Internationalor Chem-Impex) may be coupled in 5-fold excess relative to resin loadingusing DIC/Oxyma as the coupling agents and a reaction temperature ofca., 90° C. for 4 min. Fmoc-Arg(Pbf)-OH may be double coupled at 90° C.for 4 min each and Fmoc-His(Trt)-OH may be coupled using a two-stageprotocol: 4 min at rt followed by 8 min at 50° C. Single Fmocdeprotections may be carried out using 20% piperidine in DMF(deprotection solution) at 90° C. for 1.5 min.

B) Procedure for Coupling Halomethylbenzoic Acids

The Fmoc-deprotected peptide-resin (0.1 mmol) may be treated with asolution of the desired isomer (meta or para) of either chloro- orbromomethylbenzoic acid (20 eq.) and DIC (10 eq.) in DMF (4 mL) in amicrowave reactor at 75° C. for 15 min. Reaction completeness may bedetermined by the Kaiser ninhydrin test (Kaiser, et al., Anal. Biochem.,1970, 34, 595-598). In cases where the coupling is determined to beincomplete, the coupling may be repeated with fresh reagents.

C) Procedure for Peptide Cleavage from Resin

Upon completion of the SPPS, the resin may be washed extensively withDMF and then with DCM and dried. The resin may then be treated with acleavage cocktail (10 mL/0.1 mmol scale) consisting of eitherTFA/water/TIPS (95:2.5:2.5) (Cleavage Cocktail A) or more preferablywith TFA/water/phenol/TIPS (88:5:5:2) (Cleavage Cocktail B) and heatedin a microwave reactor at 38° C. for 40 min, then filtered. The resinmay be washed with TFA and the combined filtrates concentrated under astream of nitrogen to a volume of ca. 2.5 mL and the peptide may then beprecipitated by the addition of cold diethyl ether (40 mL). Thepeptide/ether suspension may be centrifuged and the ether layer wasdecanted. The peptide pellet may be re-suspended in ether, centrifugedand decanted, and this process may be repeated a third time. The crudepeptide thus obtained may then be dried under a mild nitrogen stream.

D) Procedure for Peptide Cyclization (Thioether Formation)

The crude cysteine- or homocysteine-containing peptide may be dissolvedin deoxygenated MeCN/water (50-60% MeCN) or EtOH/water (60% EtOH) at aconcentration of <4 mg/mL. The pH of the peptide solution may then beraised to ca. 7-9 through the addition of either solid NaHCO₃, sat'd aq.NaHCO₃ or 1M aq. Tris buffer (pH 7.5) and the resulting solution may bestirred at rt for 3-16 h. Typically, the cyclizations are completewithin 3-4 h, as determined by analytical LC/MS.

E) Procedure for Peptide Purification

The cyclization reaction mixture may be acidified to pH 1.5-3 by theaddition of TFA, and the solution concentrated to remove most of theorganic co-solvent (MeCN or EtOH) to a point where slight cloudingoccurs. A minimal amount of the co-solvent may be added back asnecessary to render the mixture homogeneous and the resultant solutionmay then be purified directly by preparative HPLC in multipleinjections. Purifications may be performed on either an Agilent PrepStarHPLC system or a Gilson HPLC 2020 Personal Purification System using areverse phase C18 or C8 column selected from the following: VarianPursuit XRs C18 (21×250 mm, 100 Å, 5 μm); Varian Pursuit XRs Diphenyl(30×100 mm, 100 Å, 5 μm); Zorbax 300 SB-C8 (21×250 mm, 300 Å, 5 μm);Waters Atlantis T3 C18 (19×250 mm, 100 Å, 5 μm); Agilent Polaris 5 C18-A(30×250 mm, 180 Å, 5 μm). The mobile phase may consist of gradientelutions of buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN)ranging in initial concentration of 10-20% B to final concentrations of40-90% B with run times ranging between 36-80 min. UV detection may bemonitored at 220 and 254 nm. Product-containing fractions may beanalyzed by analytical HPLC on an Agilent 1100 HPLC system using anappropriate column type from above (4.6×250 mm, 5 μm). Pure fractionsmay be combined, concentrated to remove most of the organic phase, andthen lyophilized. TFA/HCl salt exchange may be subsequently carried outby triple lyophilization from 2 mM HCl, according to the proceduredescribed by Andrushchenko, et al., (J. Pept. Sci., 2006, 13, 37-43).

A general synthetic procedure for the synthesis of C-terminal amidesNTSC-PYY peptides wherein the BRIDGE is —NHC(O)CH₂S— is shown in Scheme2.

Steps A, C, D, and purification (E) are essentially the same as thosedescribed in Scheme 1. However, an alternate BRIDGE may be introduced inStep B, as described below.

The Fmoc-deprotected peptide-resin (0.1 mmol) may be treated with asolution of bromoacetic anhydride (6-20 eq.) in DMF (5 mL) in amicrowave reactor at 50° C. for 5 min, by which time the reaction may begenerally determined to be complete as per a Kaiser ninhydrin test. Incases where the coupling is determined to be incomplete, the couplingmay be repeated with fresh reagents.

A general synthetic procedure for the synthesis of C-terminal amideNTSC-PYY peptides

wherein Z₃₅ is

is shown in Scheme 3.

A) HATU-Mediated Coupling:

In a fritted microwave reaction vessel (supplied by CEM Corporation),NovaSyn TG Sieber resin (supplied by Novabiochem) (0.1 mmol) may betreated with deprotection solution (5 mL) and heated at 50° C. for 2.5min. The reaction is drained, washed with DMF and treated again withdeprotection solution at 50° C. for 5 min. After draining and washingthe resin with DMF, a third deprotection treatment is carried out at 50°C. for 5 min. The resin is drained and washed extensively with DMF andthen with DCM. The resin is then treated with a solution ofFmoc-Arg(Pbf)-psi-(N-Boc)Tyr(tBu)-OH from Scheme 14 (3-5 eq.), HATU(2.75-4.5 eq.) and DIEA (6-10 eq.) in DMF (4 mL) and mixed at rt for 24h. The mixture is drained and the resin was washed extensively with DMF.The resin is then capped by treatment with 20% Ac₂O in DMF (5 mL) undermicrowave conditions at 50° C. for 5 min. The reaction is drained andthe resin is washed extensively with DMF and then with DCM.

A) (Alternative) DIC/Oxyma-Mediated Coupling:

In a fritted microwave reaction vessel, NovaSyn TG Sieber resin (0.1mmol) may be deprotected as described in Step A above, then treated witha solution of moc-Arg(Pbf)-psi-(N-Boc)Tyr(tBu)-OH (2.75 eq.), DIC (2.75eq.), Oxyma (2.75 eq.) and DIEA (0.275 eq.) in MeCN (4 mL) and mixed atrt for 24 h. The reaction is drained and the resin is washed extensivelywith DMF and then with DCM and may be used directly without capping.

B) Elaboration of Reduced Amide (Psi-R35,Y36) Peptide on Pre-LoadedSieber Resin

Further amino acid extensions onto the pre-loaded (psi-R35,Y36)-Sieberresin may be performed on a CEM Liberty Blue Microwave peptidesynthesizer. Standard Fmoc-protected amino acids ware coupled in 5-foldexcess relative to the initial resin loading using HBTU/DIEA as thecoupling agents and a reaction temperature of ca., 50° C. for 15 min.Fmoc-Arg(Pbf)-OH may be double coupled using a two-stage protocol foreach coupling: 25 min at rt followed by 15 min at 50° C. andFmoc-His(Trt)-OH may be double-coupled using a two-stage protocol foreach coupling: 4 min at rt followed by 8 min at 50° C. Fmocdeprotections may be carried out in two stages using fresh deprotectionsolution for each stage: 1) 50° C. for 2.5 min and 2) 50° C. for 5 min.In some cases, double couplings may be used advantageously throughout toimprove the quality of the isolated crude peptide.

Installation of the thioether BRIDGE moiety may proceed either by thebromomethylbenzoic acid coupling shown in Scheme 1 step B, with thereaction temperature of 50° C. Alternatively, a thioether BRIDGE moietymay be introduced using the bromoacetylation coupling shown in Scheme 2Step B.

Upon completion of the SPPS, the resin is washed extensively with DMFand then with DCM and dried. Cleavage of protected peptide from SieberResin may be then accomplished with a solution of 1-2% TFA in DCM (10mL/0.1 mmol scale) and mixed for ca. 10 min, then filtered. Thistreatment may be repeated 9 additional times using fresh cocktail witheach treatment. The combined filtrates are then concentrated to affordthe crude protected peptide as a yellow syrup/solid which may be useddirectly for subsequent global deprotection. The protected peptideobtained above is treated with Cleavage Cocktail B (10 mL) at rt for 2.5h and is then concentrated under a stream of nitrogen to a volume of ca.2.5 mL. The crude peptide may be precipitated by the addition of colddiethyl ether (40 mL). The peptide/ether suspension may be centrifugedand the ether layer is decanted. The peptide pellet may be re-suspendedin ether, centrifuged and decanted, and this process may be repeated athird time. The crude peptide thus obtained may be dried under a mildnitrogen stream. Cyclization and purification of the reduced amide(psi-R35,Y36) peptides may be accomplished according to the proceduresdescribed in Scheme 1 Step D and Step E.

Synthesis of lipidated c-terminal amide NTSC-PYY peptides may beaccomplished according to Scheme 4.

A) Procedure for Introducing Derivatized Lysine Residues into PeptideSequences Built on Standard Rink Amide Resin

To a resin-bound C-terminal amide peptide, elaborated to the pointpreceding the desired point of derivatization and prepared as describedin Scheme 1, Step A, may be sequentially coupled either Dde-Lys(Fmoc)-OHor ivDde-Lys(Fmoc)-OH and then Fmoc-Glu-OtBu (all supplied byNovabiochem) under microwave conditions (either manually or on theLiberty Blue Peptide Synthesizer) using DIC/Oxyma coupling methods asdescribed in Scheme 1, Step A. Following Fmoc deprotection, the resinmay be treated with a solution of the lipophilic acid [palmitic acid orα-tocopheryloxyacetic acid (AcVitE)] (5-10 eq.), DIC (5-10 eq.) andeither HOBT or Oxyma (5-10 eq.) in DMF under microwave conditions at 90°C. for 10 min. The reaction is then drained and the resin is washed withDMF.

B) Procedure for Deprotecting Dde- or ivDde-Protected Lysinyl Peptide

The derivatized lysinyl peptide resin may be treated with a solution of3% hydrazine in DMF (6 mL/0.1 mmol resin) under microwave conditions at90° C. for 3.5 min. The reaction is drained and this procedure may berepeated two additional times. The reaction is drained and the resin iswashed extensively with DMF and then with DCM.

C) Procedure for Direct Incorporation of Fmoc-Lys(Pal-Glu-OtBu)-OHResidue

In cases where the palmitoylated-γ-Glu-Lysinyl residue is to beincorporated into the sequence, Fmoc-Lys(Pal-Glu-OtBu)-OH (availablefrom Peptides International or ActivePeptide) may be used directly inthe procedure described in Scheme 1, Step A.

Compounds of the present invention with lipdated lysine residues may becompleted by following the procedures of Scheme 1, Steps B, C, D, and E.

Synthesis of lipidated, reduced amide (psi-R35,Y36), c-terminal amide,NTSC-PYY peptides may be accomplished according to Scheme 5.

A) Procedure for Introducing Derivatized Lysine Residues into PeptideSequences Built on (Psi-R35,Y36) Pre-Loaded Sieber Resin

To a partially elaborated reduced amide (psi-R35,Y36) peptide, preparedas described in Scheme 3, Step A, may be sequentially coupledAlloc-Lys(Fmoc)-OH (available from Chem-Impex or AAPPTec, LLC) followedby Fmoc-Glu-OtBu and then palmitic acid (each at 5 eq., using eitherHATU/DIEA or HBTU/DIEA coupling methods at 50° C. for 15 min. When thelipophilic acid to be coupled is stearic acid, arachidic acid,octadecanedioic acid, mono-tert-butyl ester (available from AstaTech,Inc.) or 20-(tert-butoxy)-20-oxoicosanoic acid (available from KeyOrganics, Inc.), NMP may be used as solvent for reasons of improvedreagent solubilities, and the coupling reaction may be mediated byHATU/DIEA at 50° C. for 30 min. Alternatively for arachidic acid, thecoupling could be carried out using the DIC/Oxyma-mediated proceduredescribed in Scheme 1, Step A, but with reagents at 5 eq. and THF as thereaction solvent.

B) Procedure for Alloc Deprotection

The above resin (0.1 mmol) is washed with deoxygenated DCM and thentreated with PhSiH₃ (12.5 eq.) in deoxygenated DCM (5 mL). After 2 min,a solution of Pd(PPh₃)₄(0.25 eq) in deoxygenated DCM (5 mL) may be addedand the reaction is mixed for 0.5 h under a nitrogen atmosphere. Thereaction is drained and the resin is washed 1× with deoxygenated DCM.The resin is again treated with PhSiH₃ (12.5 eq.) and Pd(PPh₃)₄(0.25 eq)as above and reacted for an additional 0.5 h. The reaction is drainedand the resin is successively washed extensively with DCM, DMF and DCM.

Further elaboration and completion of the peptide may be performed asdescribed herein above, beginning with Scheme 3, Step B.

Synthesis of C-terminal amide NTSC-PYY analogues wherein BRIDGE istriazolyl is shown in Scheme 6.

A) Synthesis of Azido- and Akynyl-Containing Resin-Bound C-TerminalAmide Peptides

The resin-bound ε-azidonorleucine-containing protected peptide, cappedon the N-terminus with 4-pentynoic acid may be prepared according toScheme 1, Step A. A double-coupling protocol may be used for theincorporation of 4-pentynoic acid.

B) Procedure for Introducing Derivatized Lysine Residues into Azido- andAkynyl-Containing Resin-Bound C-Terminal Amide Peptides

Fmoc-Lys(Dde)-OH may be incorporated into the SPPS at the sequenceposition to be derivatized, following the procedure described in Scheme1, Step A. Upon completion of the linear sequence (following theincorporation of 4-pentnoic acid), the resin may be treated with 3%hydrazine in DMF (8 mL/0.1 mmol scale) for 5 min at rt and then themixture is drained. This procedure may be repeated ca. 6×, after whichthe resin is washed extensively with DMF and then DCM. Fmoc-Glu-OtBu andthe lipophilic acid is then sequentially coupled onto theDde-deprotected resin following the procedure described in Example 6A.

C) Procedure for Azido- and Alkynyl-Containing Peptide Cleavage fromResin

Upon completion of the SPPS, the resin may be washed extensively withDMF and then with DCM and dried. The resin is then treated with acleavage cocktail (10 mL/0.1 mmol scale) consisting ofTFA/water/DODT/TIPS (92.5:2.5:2.5:2.5) (Cleavage Cocktail C) and heatedin a microwave reactor at 38° C. for 40 min, then filtered. The resin iswashed with TFA and the combined filtrates are concentrated under astream of nitrogen to a volume of ca. 2.5 mL and the peptide may beprecipitated by the addition of cold diethyl ether (40 mL). Thepeptide/ether suspension may be centrifuged and the ether layer isdecanted. The peptide pellet may be re-suspended in ether, centrifugedand decanted, and this process may be repeated a third time. The crudepeptide thus obtained may be dried under a mild nitrogen stream.

D) Procedure for Peptide Cyclization (Triazole Formation)

The following solutions may be prepared using deoxygenated water:

1) CuSO₄ (7 mg in 2 mL of water)

2) 30 mg of TBTA in 5.4 mL of EtOH and 0.6 mL of MeCN

3) Premix solution 1 (943 μL) and solution 2 (4.8 mL)

4) sodium ascorbic acid (30 mg in 3 mL of water)

To a solution of the crude peptide from Step C (0.021 mmol) in eitherdeoxygenated water or HEPES buffer (pH 7.4) (20 mL) may be addedsolution 3, followed by solution 4 (2.4 mL), and the resultant milkysolution is warmed to 35-40° C. until cyclization is complete, asdetermined by LCMS analysis (ca., 1-5 h). The reaction solution may bediluted to 40 mL with either water (0.1% TFA) or 60% MeCN/water (0.1%TFA), filtered and purified directly by preparative HPLC using multipleinjections as described in Scheme 1, Step E.

Synthesis of C-terminal amide NTSC-PYY analogues wherein BRIDGE is alactam is shown in Scheme 7.

A) Synthesis of Resin-Bound C-Terminal Amide Peptide

The protected peptidyl resin may be synthesized using an Fmoc strategyon a Symphony X peptide synthesizer from Protein Technologies. Couplingsmay be performed on either Rink amide resins or Sieber resins using HATUand NMM in DMF for 10 min at rt. Fmoc amino acids may be used in 6-foldexcess and double-coupled. For peptides containing psi-(R35,Y36)modification, Fmoc-Arg(Pbf)Ψ[CH₂N(Boc)]Tyr(t-Bu)-OH was coupled in3-fold excess using HATU and NMM in DMF for 1 hr at rt. The c-amino ofthe terminal residue of the linear sequence may be Boc-protected and theγ-carboxyl group of the glutamic acid residue that forms the lactambridge may be allyl protected. Lysine(s) in the peptide may beorthogonally Dde protected.

B) Synthesis of γ-Glutamate-N-Hydroxysuccinimide Ester

Following completion of the linear sequence the glutamate side chainallyl protecting group may be removed using Pd(PPh₃)₄ and PhSiH₃ in DCM.The N-hydroxysuccinimide (NHS) ester may be then synthesized bydouble-coupling NHS using HATU and DIEA for 10 min at RT.

C) Procedure for Lactam Cyclization

The peptide may be cleaved from the resin and globally deprotected bytreatment with TFA/TIPS/water (95:2.5:2.5) for 2 h at RT, thenprecipitated into ether and collected by centrifugation and dried. Thecrude product may be dissolved in DMSO; TEA (10 eq.) is added and thecyclization allowed to proceed for 1 h at rt. The crude product may bediluted with water and purified by preparative RP-HPLC. Orthogonallydde-protected lysines may be deprotected by dissolving the peptide in 2%hydrazine/DMF (at a concentration of 5 mM) and stirring for 1 h at RT.The reaction mixture may be diluted with water and the pH is adjusted to2 with the addition of 10% TFA/water and the solution may be directlypurified by preparative RP-HPLC as described in Scheme 1, Step E.Solution-phase synthesis of lipidated C-terminal amide NTSC-PYYanalogues wherein BRIDGE is a lactam is shown in Scheme 8.

Solution phase synthesis of lipidated C-terminal amide NTSC-PYYanalogues wherein BRIDGE is a lactam is shown in Scheme 8.

The peptide obtained in Scheme 7 above, wherein only one of Z₄, Z₇, Z₉,Z₁₁, Z₂₂, Z₂₃, or Z₃O may be lysine, is dissolved in DMF (at aconcentration of 5 mM), TEA (5 eq.) is added, followed by the N-hydroxyester of the protected lipid (2 eq). The reaction may be allowed toproceed overnight at rt and then purified by preparative HPLC. Thet-butyl esters may be deprotected in TFA/TIPS/water (95:2.5:2.5) for 30min at rt and the reaction concentrated and purified by preparativeRP-HPLC as described in Scheme 1, Step E.

General Procedures for Synthesis of Bromoacetylated Cyclic-ThioetherPeptides

Schemes 9, 10, and 11 all show various routes to bromoacetylatedcyclic-thioether peptides of the present invention. In Scheme 9, thethioether bridge is formed by reaction of a thiol-containing amino acidside chain in the peptide sequence with a bromoacetyl group at the aminoterminus of the peptide. Scheme 10 illustrates a similar approach asScheme 9 but with a discrete PEG spacer inserted between the lysine sidechain and the bromoacetyl group. Scheme 11 illustrates a strategy forsynthesis of another class of bromoacetylated cyclic-thioether peptidesin which the bridge is formed by reaction of a thiol nucleophile at theamino terminus of the peptide with a bromoacetyl group covalentlyattached to a lysine side chain within the peptide sequence.

A. Synthesis of Resin-Bound C-Terminal Amide Peptide

The protected peptide on resin may be synthesized on Sieber amide orRink amide resins using FMOC strategy. Standard FMOC-protected aminoacids (supplied by Novabiochem (EMD Millipore), Bachem, PeptidesInternational or Chem-Impex) may be coupled in 3-6-fold excess relativeto resin loading using DIC/Oxyma, HBTU/DIPEA or HATU/NMM as the couplingagents at room temperature or elevated temperature. Double-couplingmight be carried out for superior purity, and especially for the aminoacid coupled onto the ca-N-alkylated amino acids. For peptidescontaining psi-(R35,Y36) modification,Fmoc-Arg(Pbf)Ψ[CH₂N(Boc)]Tyr(t-Bu)-OH may be coupled in 3-fold excessusing HATU and NMM in DMF for 1 hr at rt.

B. Procedure for Bromoacetylation of Resin-Bound Peptide

The lysine to be bromoacetylated may be orthogonally protected witheither an alloc, ivDDE or DDE protecting group. Following completion ofthe linear sequence on resin the orthogonally protected lysine may bedeprotected (for alloc, Pd(Ph₃)₄ and phenyl silane in DCM; for DDE orivDDE, 2% hydrazine in DMF) and the amino group may be bromoacetylatedunder various conditions such as, 1) reacting with a large excess ofbromoacetic anhydride in DMF in a microwave reactor at 50° C. for 5 min,by which time the reaction may be generally determined to be complete asper a Kaiser ninhydrin test; 2) reacting with a large excess ofbromoacetic anhydride in DMF or DCM in the presence of base such as TEAor DIPEA at room temperature; or 3) coupling with bromoacetic acid usingDIC, or DIC/Oxyma.

C. Procedure for Peptide Cleavage from Resin

Upon completion of the SPPS, the resin may be washed extensively withDMF and then with DCM and dried. With Sieber amide resin-bound peptide,the dried resin may be treated with a solution of 1 to 2% TFA in DCM (10mL) for 5 to 10 min, then filtered. This treatment may be repeated a fewmore times using fresh cocktail for each treatment. The filtrates arethen combined and concentrated to afford the crude protected peptide asa yellow foam. This foam may be then treated with cleavage cocktailTFA/phenol/H₂O/TIPS (88/5/5/2), or TFA/water/TIPS (95:2.5:2.5), orTFA/phenol/H₂O/TIPS/DTT (84/10/2.5/2.5/1), and heated in a microwavereactor at 38° C. for 30-45 min, or at room temperature for 2-3.5h. Thecrude peptide may be precipitated in cold diethyl ether. Thepeptide/ether suspension may be centrifuged and the ether layer isdecanted. The peptide pellet may be re-suspended in ether, centrifugedand decanted, and this process may be repeated a third time. The crudepeptide thus obtained may be dried under a mild nitrogen stream.

Alternatively, the Sieber amide or Rink amide resin-bound peptide may betreated with cleavage cocktail as above without prior treatment with1-2% TFA in DCM to afford the fully deprotected peptide.

D. Procedure for Peptide Cyclization (Thioether Formation)

The crude free thiol and bromoacetamide-containing peptide may bedissolved in deoxygenated MeCN/water or EtOH/water at a concentration of4 to 10 mg/mL with optional addition of EDTA. The pH of the peptidesolution may be then raised to ca. 7-9 through the addition of base suchas NaHCO₃, NaOH, DIPEA, or TEA and the resulting solution is stirred atroom temperature for 0.25-2.5 h.

Alternatively, the crude peptide could be purified by HPLC, the peptidefractions combined and basified to about pH 7-9, and stirred at roomtemperature optionally in the presence of EDTA for 0.25 to 2.5h. Afteracidification, the reaction solution may be concentrated at roomtemperature to remove organic solvent, and then subjected to HPLCpurification.

E. Procedure for Peptide Purification

The cyclization reaction mixture may be acidified with TFA, and thesolution is concentrated to remove most of the organic co-solvent (MeCNor EtOH), and the resultant solution may be then purified directly bypreparative HPLC on a reversed-phase column. The mobile phase consistsof gradient elutions of buffer A (0.1% TFA in water) and Buffer B (0.1%TFA in MeCN) ranging in initial concentration of 0-20% B to finalconcentrations of 40-90% B with run times ranging between 20-60 min. UVdetection may be monitored at 220 and 254 nm. Product-containingfractions may be analyzed by HPLC on an Agilent 1100 HPLC system using aWaters T3 Atlantis C18 column (4.6×250 mm, 5 μm). Pure fractions may becombined, concentrated to remove most of the organic phase, and thenlyophilized.

General Procedures for Synthesis of Bromoacetylated Cyclic-LactamPeptides

The cyclic lactam peptides may be synthesized according to theprocedures shown in Scheme 12. Cyclic lactam peptide is firstsynthesized according to Scheme 7 wherein only one of Z₄, Z₇, Z₉, Z₁₁,Z₂₂, Z₂₃, or Z₃₀ is lysine. The lysine is then bromoacetylated usingbromoacetic acid N-hydroxysuccinimide ester (3-7 eq) in 10% ACN/water atpH 10, RT, 20 mins. The final bromoacetylated peptide may be purified byRP-HPLC as outlined for the cyclic-thioether peptides.

General Procedures for Synthesis of Bromoacetylated CyclicTriazole-Linked Peptides

The bromoacetylated cyclic triazole-linked peptides may be synthesizedaccording to the procedure shown in Scheme 13. The linear protectedpeptide on resin may be synthesized in a similar manner as described forthe cyclic-thioether peptides in Scheme 10 except that L-azido-lysinemay be incorporated for triazole formation and the N-terminal residuemay be 4-pentynoic acid. The Fmoc may be removed with 20% piperidine inDMF and then reacted with bromoacetic anhydride. The linear sequence maybe globally deprotected (TFA/TIPS/water:95%/2.5%/2.5%) and the crudepeptide precipitated into cold ether, collected by centrifugation, andpurified by preparative RP-HPLC. The purified linear peptide may becyclized in the presence of CuSO₄/TBTA and NaASrb in buffer solution(HEPES, MOPS, etc.) to give cyclic triazole-linked peptides, which maybe purified by preparative RP-HPLC as described in Scheme 1, step E.

An alternative class of cyclic triazole-linked peptides can synthesizedby persons skilled in the art beginning with a linear sequence in whichthe N-terminal residue is an azido carboxylic acid (for example, 5-azidopentanoic acid) and the residue in position 30 or 31 is an alkynyl aminoacid (for example, 2-amino-7-octynoic acid) in a similar fashion asdescribed above.

Peptide Analysis and Characterization

Purified peptides were analyzed by LC/MS on a Hewlett Packard Series1100 MSD system configured with an HP 1100 series HPLC using a WatersAtlantis T3 C18 (4.6×250 mm, 300 Å, 5 μm) column. Depending on thepolar/non-polar nature of the peptide, one of three gradients was used(buffers A and B as above) at a flow rate of 1 mL/min and a columntemperature of 35° C.: Method 1) 15-60% B over 22 min; Method 2) 30-60%B over 22 min; Method 3) 40-90% B over 22 min. Electrospray analysis(ES-API, positive ion scan) provided mass analysis for each peptide. Inall cases, multiple charged species were observed with 1/3[M+3]⁺ and1/4[M+4]⁺ ions being the characteristic, most prominently observed ions.All products yielded their expected multi-charged ions within acceptablelimits. Results of the mass spectral analyses of the peptides andobserved LC retention times (RT) are shown in Table 1:

TABLE 1 AnalytiCal data for NTSC-PYY CompoundS. Seq. I.D. 1/3[M + 3]⁺1/3[M + 3]⁺ 1/4[M + 4]⁺ 1/4M + 4]⁺ HPLC RT No. Mol. Formula MW (Calc'd)(Found) (Calc'd) (Found) Meth. (min) 1 C₂₁₅H₃₄₅BrN₅₆O₆₇S 4898.41 1633.801633.8 1225.60 1225.4 1 12.48 2 C₁₈₇H₂₈₁N₅₃O₅₅S 4183.66 1395.55 1395.41046.92 1046.6 1 11.7 3 C₁₈₆H₂₈₄N₅₆O₅₅ 4184.63 1395.88 1395.6 1047.161047.0 1 11.1 4 C₂₁₀H₃₂₅N₅₃O₅₇S 4564.27 1522.42 1522.2 1142.07 1141.9 217.0 5 C₂₂₅H₃₄₅N₅₅O₅₉S 4796.59 1599.86 1599.6 1200.15 1199.9 3 15.8 6C₂₂₄H₃₄₈N₅₈O₅₉ 4797.56 1600.19 1600.1 1200.39 1200.3 3 15.7 7C₂₁₂H₃₂₇N₅₅O₅₉S 4622.31 1541.77 1541.8 1156.58 1156.7 2 15.8 8C₂₀₈H₃₁₉N₅₅O₅₉S 4566.20 1523.07 1522.8 1142.55 1142.4 2 14.7 9C₁₈₇H₂₈₃N₅₃O₅₄S 4169.68 1390.89 1390.6 1043.42 1043.3 1 11.3 10C₂₂₃H₃₃₉N₅₅O₆₁S 4798.52 1600.51 1600.4 1200.63 1200.6 3 13.4 11C₂₂₃H₃₄₁N₅₅O₆₀S 4784.54 1595.85 1595.7 1197.14 1197.1 3 12.8 12C₁₈₈H₂₈₃N₅₃O₅₅S 4197.69 1400.23 1400.2 1050.42 1050.3 1 11.9 13C₂₀₈H₃₂₁N₅₅O₅₈S 4552.22 1518.41 1518.2 1139.06 1139.0 2 13.8 14C₂₀₉H₃₂₁N₅₅O₅₉S 4580.23 1527.74 1527.8 1146.06 1145.9 2 15.2 15C₁₉₉H₃₀₉N₅₅O₅₉S 4448.02 1483.67 1483.7 1113.01 1112.9 2 14.1 16C₂₀₂H₃₀₈N₅₄O₅₈S 4453.04 1485.35 1485.1 1114.26 1114.2 2 13.9 17C₂₀₅H₃₁₂N₅₄O₅₉S 4509.10 1504.03 1503.9 1128.28 1128.2 2 16.6 18C₂₀₇H₃₁₄N₅₄O₆₁S 4567.14 1523.38 1523.2 1142.79 1142.7 2 16.4 19C₂₀₈H₃₁₉N₅₅O₅₉S 4566.20 1523.07 1523.1 1142.55 1142.5 2 15.0 20C₂₀₈H₃₁₉N₅₅O₅₉S 4566.20 1523.07 1522.8 1142.55 1142.4 2 15.1 21C₂₀₂H₃₁₀N₅₂O₅₉S 4443.04 1482.01 1481.9 1111.76 1111.6 2 17.2 22C₂₀₄H₃₁₂N₅₂O₆₁S 4501.08 1501.36 1501.2 1126.27 1126.2 2 16.8 23C₂₂₂H₃₄₅N₅₇O₆₆S 4900.57 1634.52 1634.4 1226.14 1225.9 2 16.7 24C₂₂₄H₃₄₉N₅₇O₆₆S 4928.62 1643.87 1643.4 1233.16 1233.0 2 18.4 25C₂₁₀H₃₂₅N₅₅O₅₈S 4580.27 1527.76 1527.7 1146.07 1146.1 2 17.0 26C₂₁₂H₃₂₉N₅₅O₅₈S 4608.32 1537.11 1537.1 1153.08 1153.0 2 19.7 27C₂₁₇H₃₃₈N₅₆O₆₃S 4771.45 1591.48 1591.3 1193.86 1193.8 2 12.0 28C₂₀₂H₃₁₀N₅₄O₅₇S 4439.06 1480.69 1480.6 1110.77 1110.8 2 13.3 29C₁₉₉H₃₁₁N₅₅O₅₈S 4434.04 1479.01 1478.9 1109.51 1109.3 2 13.7 30C₁₉₉H₃₁₁N₅₅O₅₈S 4434.04 1479.01 1479.0 1109.51 1109.3 2 14.2 31C₂₂₂H₃₄₇N₅₇O₆₄S 4870.58 1624.53 1624.6 1218.65 1218.6 2 18.5 32C₂₀₅H₃₁₄N₅₄O₅₈S 4495.12 1499.37 1499.3 1124.78 1124.8 2 15.9 33C₂₀₉H₃₂₃N₅₅O₅₈S 4566.24 1523.08 1522.9 1142.56 1142.4 2 14.6 34C₂₀₁H₃₁₅N₅₅O₅₈S 4462.09 1488.36 1488.0 1116.52 1116.4 2 13.9 35C₂₀₉H₃₂₃N₅₅O₅₈S 4566.24 1523.08 1522.8 1142.56 1142.5 2 14.4 36C₂₀₀H₃₁₃N₅₅O₅₈S 4448.07 1483.69 1483.5 1113.02 1112.9 2 13.8 37C₂₀₅H₃₂₂N₅₆O₅₉S 4547.20 1516.73 1516.6 1137.80 1137.7 2 14.2 38C₂₀₆H₃₂₄N₅₆O₅₉S 4561.22 1521.41 1521.7 1141.31 1141.3 2 13.7 39C₂₀₉H₃₂₂N₅₄O₅₉S 4567.23 1523.41 1523.2 1142.81 1142.7 3 10.3 40C₂₁₉H₃₄₆N₆₀O₆₄ 4843.52 1615.50 1615.4 1211.9 1211.8 2 15.1 41C₂₀₇H₃₂₄N₅₈O₅₈ 4553.20 1518.70 1518.4 1139.3 1139.3 2 13.6 42C₂₂₁H₃₄₈N₆₀O₆₆ 4901.56 1634.90 1634.8 1226.4 1226.4 2 11.0 43C₂₂₀H₃₄₃N₅₇O₆₄S 4842.53 1615.18 1615.0 1211.63 1211.5 2 15.5 44C₂₂₄H₃₅₁N₅₇O₆₄S 4898.64 1633.88 1633.9 1225.66 1225.6 2 12.7 45C₂₆₁H₄₂₈N₅₆O₈₁S 5678.63 1893.88 1893.4 1420.66 1420.6 2 19.4 46C₂₀₅H₃₂₂N₅₆O₅₉S 4547.20 1516.73 1516.6 1137.80 1137.6 2 13.7 47C₂₂₅H₃₅₁N₅₇O₆₆S 4942.65 1648.55 1648.2 1236.66 1236.6 2 11.7 48C₂₂₅H₃₅₁N₅₇O₆₆S 4942.65 1648.55 1648.6 1236.66 1236.6 2 11.8 49C₂₀₈H₃₂₁N₅₅O₅₉S 4568.22 1523.74 1523.6 1143.06 1142.9 1 15.2 50C₂₂₅H₃₅₁N₅₇O₆₅S 4926.65 1643.22 1643.2 1232.66 1232.5 2 11.6 51C₂₀₅H₃₁₄N₅₄O₅₉S 4511.12 1504.71 1504.6 1128.78 1128.6 2 15.6 52C₂₃₇H₃₈₀N₅₆O₆₉S 5150.00 1717.67 1717.5 1288.50 1288.4 2 18.6 53C₂₃₄H₃₇₁N₅₉O₆₈S 5130.92 1711.31 1710.9 1283.73 1283.6 2 16.6 54C₂₂₂H₃₄₉N₅₇O₆₂S 4840.60 1614.53 1614.3 1211.15 1211.1 2 16.6 55C₂₂₃H₃₄₉N₅₇O₆₃S 4868.61 1623.87 1623.6 1218.15 1218.0 2 14.9 56C₂₀₆H₃₀₀ClN₅₅O₅₈S 4542.48 1515.16 1515.0 1136.62 1136.4 1 13.6 57C₂₀₇H₃₀₁Cl₂N₅₅O₅₉S 4606.95 1536.65 1536.3 1152.74 1152.5 1 14.5 58C₂₀₉H₃₁₄FN₅₅O₅₈S 4576.17 1526.39 1526.2 1145.04 1144.9 2 10.9 59C₂₂₃H₃₄₉N₅₇O₆₄S 4884.61 1629.20 1629.0 1222.15 1221.9 2 15.5 60C₂₂₃H₃₄₉N₅₇O₆₄S 4884.61 1629.20 1629.1 1222.15 1222.1 2 15.4 61C₂₁₀H₃₁₄F₃N₅₅O₅₈S 4626.18 1543.06 1542.7 1157.55 1157.4 2 12.7 62C₂₀₄H₃₁₀F₃N₅₅O₅₈S 4550.08 1517.69 1517.6 1138.52 1138.3 2 9.1 63C₂₀₇H₃₁₆F₃N₅₅O₅₈S 4592.16 1531.72 1531.7 1149.04 1149.0 2 12.1 64C₂₁₀H₃₂₅N₅₅O₅₉S 4596.27 1533.09 1532.7 1150.07 1150.0 2 11.8 65C₂₀₈H₃₁₂D₉N₅₅O₅₈S 4561.15 1521.38 1521.3 1141.29 1141.2 2 14.1 66C₂₁₁H₃₁₃F₆N₅₅O₅₈S 4694.17 1565.72 1565.4 1174.54 1174.6 2 14.1 67C₂₁₁H₃₁₃F₆N₅₅O₅₈S 4694.17 1565.72 1565.6 1174.54 1174.4 2 14.2 68C₂₁₉H₃₄₈N₅₈O₆₅S 4865.57 1622.86 1622.7 1217.39 1217.3 1 17.3 69C₁₈₁H₂₈₀N₅₄O₅₄ 4076.53 1359.84 1359.7 1020.13 1020 1 11.4 70C₁₈₀H₂₇₈N₅₄O₅₄ 4062.50 1355.17 1355 1016.63 1016.7 1 12.2 71C₁₈₁H₂₇₈N₅₄O₅₅ 4090.51 1364.50 1364.4 1023.63 1023.5 1 12.8 72C₂₁₅H₃₃₉N₅₇O₆₆ 4778.38 1593.79 1593.4 1195.60 1195.6 1 15.8 73C₂₀₃H₃₂₁BrN₅₆O₆₁S 4634.09 1545.70 1545.4 1159.52 1159.6 1 12.12 74C₁₈₈H₂₉₂BrN₅₅O₅₄S 4298.69 1433.90 1433.9 1075.67 1075.6 1 11.96 75C₂₁₁H₃₃₈BrN₅₅O₆₆S 4813.30 1605.43 1605.5 1204.32 1204.4 1 12.27 76C₂₄₉H₄₁₂BrN₅₇O₈₄S 5660.31 1887.77 1887.4 1416.08 1416.0 1 13.15 77C₂₁₄H₃₄₃BrN₅₆O₆₇S 4884.38 1629.13 1629.1 1222.09 1222.1 1 12.24 78C₂₁₆H₃₄₇BrN₅₆O₆₇S 4912.44 1638.48 1638.4 1229.11 1229.1 1 12.49 79C₂₁₆H₃₄₇BrN₅₆O₆₇S 4912.44 1638.48 1638.1 1229.11 1228.9 1 12.58 80C₂₂₀H₃₄₄BrN₅₉O₆₇S 4999.48 1667.49 1667.3 1250.87 1250.8 1 12.55 81C₂₂₀H₃₄₄BrN₅₉O₆₇S 4999.48 1667.49 1667.4 1250.87 1250.7 1 12.43 82C₂₁₁H₃₃₈BrN₅₅O₆₈S 4845.30 1616.10 1616.0 1212.32 1212.4 1 12.44 83C₂₁₇H₃₄₇BrN₅₈O₆₆ 4904.39 1635.80 1635.9 1227.10 1227.0 1 12.55 84C₂₂₅H₃₅₄BrN₅₉O₆₆S 5053.61 1685.54 1685.4 1264.40 1264.3 1 12.80 85C₂₁₆H₃₄₅BrN₅₆O₆₈S 4926.42 1643.14 1642.9 1232.61 1232.6 1 11.72 86C₂₁₆H₃₄₅BrN₅₆O₆₉S 4942.42 1648.47 1648.3 1236.61 1236.5 1 11.92 87C₂₁₆H₃₄₅BrN₅₆O₆₉S 4942.42 1648.47 1648.3 1236.61 1236.4 1 11.82 88C₂₁₂H₃₃₉BrN₅₆O₆₇S 4856.33 1619.78 1619.5 1215.08 1214.9 1 12.43 89C₂₁₃H₃₃₉BrN₅₆O₆₈S 4884.32 1629.11 1629.0 1222.08 1222.0 1 ND 90C₂₁₄H₃₄₁BrN₅₆O₆₈S 4898.32 1633.77 1633.7 1225.58 1225.5 1 ND 91C₂₁₄H₃₄₁BrN₅₆O₆₈S 4898.32 1633.77 1633.3 1225.58 1225.5 1 ND 92C₂₁₆H₃₄₅BrN₅₆O₆₈S 4926.42 1643.14 1642.7 1232.61 1232.6 1 ND 93C₂₁₃H₃₄₁BrN₅₆O₆₇S 4870.35 1624.3 1624.1 1218.5 1218.4 1 12.67 94C₂₁₃H₃₄₁BrN₅₆O₆₇S 4870.35 1624.3 1624.3 1218.5 1218.4 1 12.42 95C₁₈₂H₂₇₉BrN₅₄O₅₅ 4183.45 1395.5 1395.3 1046.9 1046.7 1 13.41 96C₂₃₉H₃₉₃BrN₅₆O₇₉S 5427.04 1810.0 1809.7 1357.8 1357.8 1 12.44 97C₂₁₄H₃₄₃BrN₅₆O₆₇S 4884.4 1629.1 1628.8 1222.1 1221.9 1 11.74 98C₁₈₆H₂₈₆BrN₅₅O₅₅S 4284.6 1429.2 1428.9 1072.1 1071.9 1 12.28 99C₁₈₇H₂₈₈BrN₅₅O₅₅S 4298.6 1433.9 1433.6 1075.6 1075.6 1 12.03 100C₁₈₆H₂₈₈BrN₅₅O₅₄S 4270.6 1424.5 1424.3 1068.6 1068.6 1 12.59

Intermediates

It is understood that the following examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggestive to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporate byreference in their entirety for all purposes.

Intermediate 1 Synthesis of α-Tocopheryloxyacetic Acid (AcVitE) (8) A)tert-Butyl α-tocopheryloxyacetate (7)

A mixture of α-tocopherol (1.14 g, 2.65 mmol), tert-butyl bromoacetate(470 μL, 3.18 mmol) and K₂CO₃ (1.1 g, 7.94 mmol) in acetone (10 mL) wasstirred at rt for 2-3 d. The mixture was then filtered through a smallplug of K₂CO₃ and the filtrate was concentrated under reduced pressure.The resultant residue was purified by silica gel chromatography elutingwith EtOAc/heptanes (0-5%) to afford tert-butyl α-tocopheryloxyacetate(7) as a colorless oil. ¹H NMR (CDCl₃) δ 4.17 (s, 2H), 2.56 (t, J=6.57Hz, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.07 (s, 3H), 1.65-1.86 (m, 2H),1.52 (s, 9H), 0.98-1.46 (m, 22H), 0.80-0.90 (m, 14H).

B) α-Tocopheryloxyacetic Acid (AcVitE) (8)

To a solution of tert-butyl α-tocopheryloxyacetate (7) (1.4 g, 2.57mmol) in DCM (12 mL) was added TFA (6 mL), and the resulting solutionwas stirred at rt. After 2 h, the solution was concentrated underreduced pressure and the resultant dark oil was purified by silica gelchromatography eluting with MeOH/DCM (0-2.5% containing 0.5% HOAc) toafford ca-tocopheryloxyacetic acid (AcVitE) (8) as an amber-coloredsyrup which slowly solidified under vacuum. ¹H NMR (CDCl₃) δ 4.34 (s,2H), 2.57 (t, J=6.82 Hz, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H),1.79 (qt, J=6.76, 13.01 Hz, 2H), 1.48-1.59 (m, 3H), 1.18-1.48 (m, 12H),1.01-1.17 (m, 7H), 0.77-0.93 (m, 14H). LC/MS: mass calcd. for C₃₁H₅₂O₄:488.75; found: 489.5 [M+H]⁺.

Intermediate 2 1. Synthesis of(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicAcid (16) A) Benzyl2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oate (9)

Into a 100-mL round-bottom flask, was placed2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (18 g, 68.366mmol), benzyl bromide (23.386 g, 136.732 mmol), potassium carbonate(28.346 g, 205.099 mmol) and DMF (200 mL). The resulting solution wasstirred at rt overnight and diluted with EtOAc (500 mL). The mixture waswashed with water (300 mL) and brine (150 mL×2). The organic phase wasevaporated and purified with silica gel column (EtOAc/propyl ether, 1:5)to give benzyl 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oate ascolorless oil (9). LC/MS: mass calcd. for C₁₈H₂₇NO₆: 353.2, found:354.05 [M+H]⁺.

B) Benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate (10)

Into a 500-mL round-bottom flask, was placed benzyl2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oate (12 g, 33.955mmol), TFA (19.358 g, 169.774 mmol) and DCM (150 mL). The resultingsolution was stirred at rt overnight. The mixture was evaporated anddried to give benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate as light-yellowoil (10). LC/MS: mass calcd. for C₁₃H₁₉NO₄: 253.13, found: 254.05[M+H]⁺.

C). Benzyl2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14-diazadocosan-22-oate(11)

Into a 250-mL round-bottom flask, was placed2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (8.835 g,33.558 mmol), benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate (8.5 g, 33.558mmol), HATU (15.312 g, 40.270 mmol), DIEA (8.674 g, 67.116 mmol) and DMF(100 mL). The resulting solution was stirred at rt overnight and dilutedwith EtOAc (500 mL). The organic phase was washed with water (200 mL)and brine (100 mL×2). The organic phase was evaporated and purified withsilica gel column (DCM/MeOH, 10:1) to give benzyl2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14-diazadocosan-22-oateas light-yellow oil (11). LC/MS: mass calcd. for C₂₄H₃₈N₂O₉: 498.26,found: 499.50 [M+H]⁺.

D) Benzyl 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecan-1-oate (12)

Into a 500-mL round-bottom flask, was placed benzyl2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14-diazadocosan-22-oate(15 g, 30.086 mmol), TFA (17.153 g, 150.431 mmol) and DCM (200 mL). Theresulting solution was stirred at rt overnight. The mixture wasevaporated and dried to give benzyl17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecan-1-oate aslight-yellow oil (12). LC/MS: mass calcd. for C₁₉H₃₀N₂O₇: 398.21, found:399.2 [M+H]⁺.

E) (S)-1-Benzyl 23-tert-butyl22-(((9H-fluoren-9-yl)methoxy)carbonylamino)-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate(13)

Into a 250-mL round-bottom flask, was placed benzyl17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecan-1-oate (11 g, 27.607mmol), (S)-4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-tert-butoxy-5-oxopentanoic acid (11.746 g, 27.607 mmol), HATU(12.596 g, 33.128 mmol), DIEA (7.136 g, 55.214 mmol) and DMF (100 mL).The resulting solution was stirred at rt overnight and diluted withEtOAc (500 mL). The organic phase was washed with water (200 mL×2) andbrine (200 mL). The organic phase was evaporated and purified by silicagel chromatography (DCM/MeOH, 10:1) to give (S)-1-benzyl 23-tert-butyl22-(((9H-fluoren-9-yl)methoxy)carbonylamino)-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioateas light-yellow oil (13). LC/MS: mass calcd. for C43H₅₅N₃O₁₂: 805.38,found: 806.80 [M+H]⁺.

F) (S)-1-benzyl 23-tert-butyl22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate(14)

Into a 250-mL round-bottom flask, was placed (S)-1-benzyl 23-tert-butyl22-(((9H-fluoren-9-yl)methoxy)carbonylamino)-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate(10 g, 12.408 mmol) and DBU in DCM (3%, 100 mL). The resulting solutionwas stirred at rt overnight, then washed with water (200 mL×2). Theorganic phase was concentrated. The residue was dissolved with water(200 mL) and extracted with ether (200 mL×2). The aqueous phase wasextracted with DCM (200 mL). The organic phase was evaporated and driedto give of (S)-1-benzyl 23-tert-butyl22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioateas light-yellow oil (14). LC/MS: mass calcd. for C₂₈H₄₅N₃O₁₀: 583.31,found: 584.65 [M+H]⁺.

G) (S)-1-benzyl 21,39-di-tert-butyl9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonatriacontane-1,21,39-tricarboxylate(15)

Into a 50-mL round-bottom flask, was placed (S)-1-benzyl 23-tert-butyl22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosane-1,23-dioate(1.575 g, 2.699 mmol), 18-tert-butoxy-18-oxooctadecanoic acid (1 g,2.699 mmol), HATU (1.231 g, 3.239 mmol), DIEA (697.654 mg, 5.398 mmol, 2equiv) and DMF (15 mL). The resulting solution was stirred at rtovernight and diluted with EtOAc (200 mL). The organic phase was washedwith water (100 mL×2) and brine (100 mL). The organic phase wasconcentrated and purified by silica gel chromatography (DCM/MeOH, 10:1)to give (S)-1-benzyl 21,39-di-tert-butyl9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonatriacontane-1,21,39-tricarboxylateas light-yellow oil (15). LC/MS: mass calcd. for C₅₀H₈₅N₃O₁₃: 935.61,found: 936.6 [M+H]⁺.

H)(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicacid (16)

Into a 100-mL round-bottom flask, was placed (S)-1-benzyl21,39-di-tert-butyl9,18,23-trioxo-2,5,11,14-tetraoxa-8,17,22-triazanonatriacontane-1,21,39-tricarboxylate(2.5 g, 3.041 mmol), Pd/C (10% wt, 500 mg) and MeOH (50 mL). Theresulting solution was stirred at rt overnight under H₂ (3.5 atm). Theresidue was filtered, concentrated and purified by reverse phase silicagel chromatography (NH₄HCO₃/H₂O, 0.05%) to give(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicacid as a light-yellow semi-solid (16). LC/MS: mass calcd. forC₄₃H₇₉N₃O₁₃: 845.56, found: 846.55 [M+H]⁺. ¹H NMR (300 MHz, CD3OD) δ:4.24-4.29 (m, 1H), 4.07 (s, 2H), 4.03 (s, 2H), 3.69-3.72 (m, 8H),3.57-3.67 (m, 4H), 3.45-3.49 (m, 2H), 3.34-3.42 (m, 2H), 2.23-2.35 (m,6H), 2.12-2.21 (m, 1H), 1.93-1.96 (m, 1H), 1.52-1.70 (m, 4H), 1.45-1.51(m, 18H), 1.33 (s, 24H).

EXAMPLES

Compounds of the present invention can be prepared by methods known tothose who are skilled in the art. The following examples are only meantto represent examples of the invention and are in no way meant to be alimit of the invention.

Example 1: Synthesis of Cyclic PYY Analog SEQ ID NO:1

1. Synthesis of Fmoc-psi-[Arg(Pbf)-(N-Boc)Tyr(tBu)]-OH A. Synthesis ofH₂N-Tyr(tBu)-OAll

To an ice-cooled solution of Fmoc-Tyr(tBu)-O (69 g, 150.15 mmol) andK₂CO₃ (62 g, 445.36 mmol) in DMF (500 mL) was added allylbromide (72 g,595.16 mmol), and the resultant mixture was stirred for 3 h. Ice/water(1 L) was then added and the mixture was extracted with EtOAc. Thecombined organic extracts were dried (Na₂SO₄) and concentrated underreduced pressure to afford Fmoc-Tyr(tBu)-OAll as a yellow oil. To anice-cooled solution of Fmoc-Tyr(tBu)-OAll (70 g, 140.1 mmol) in DMF (600mL) was added piperidine (150 mL) in drop-wise fashion over a period of20 min. After 3 h the reaction solution was poured into water/ice (1 L),and extracted with EtOAc (2×2 L). The combined organic extracts weredried (Na₂SO₄) and concentrated under reduced pressure. The residue thusobtained was purified by silica gel chromatography, eluting withEtOAc/petroleum ether (10:1) to afford 34 g of H₂N-Tyr(tBu)-OAll as ayellow oil.

B. Synthesis of Fmoc-Arg(Pbf)-N(Me)OMe (2)

To an ice-cooled mixture of Fmoc-Arg(Pbf)-OH (1) (64.8 g, 99.88 mmol),N,O-dimethylhydroxylamine hydrochloride (20 g, 206.2 mmol) and HATU (57g, 149.91 mmol) in DCM (500 mL) was added DIEA (52 g, 402.2 mmol) indrop-wise fashion over a period of 10 min, and the resulting mixture wasallowed to stir at room temperature overnight. The reaction was thenpoured into water/ice (1 L) and extracted with DCM (1 L). The organicextracts were dried (Na₂SO₄) and concentrated under reduced pressure toafford 70 g of crude Fmoc-Arg(Pbf)-N(Me)OMe (2) as a yellow solid, whichwas used without further purification.

C. Synthesis of Fmoc-Arg(Pbf)-CHO (3)

To a cooled (−78° C.) solution of LAH in THF (1M, 107 mL, 0.107 mmol)under an inert atmosphere of nitrogen was added through a cannula acooled (−50° C.) solution of Fmoc-Arg(Pbf)-N(Me)OMe (2) (50 g, 72.3mmol) in THF (100 mL) in a drop-wise fashion over a period of 1 h. Afterstirring at −78° C. for 5 h, the mixture was poured into 1N HCl solution(300 mL), and additional 1N HCl was added as necessary to adjust the pHto 4, and then extracted with EtOAc (2×2 L). The combined organicextracts were dried (Na₂SO₄) and concentrated under reduced pressure toafford 45 g of crude Fmoc-Arg(Pbf)-CHO (3) as a yellow solid, which wasused without further purification.

D. Synthesis of Fmoc-psi-[Arg(Pbf)-Tyr(tBu)]-OAll (4)

To an ice-cooled solution of Fmoc-Arg(Pbf)-CHO (3) from step C (45 g,71.12 mmol) and H₂N-Tyr(tBu)-OAll from step A (32 g, 115.37 mmol) in THF(200 mL), MeOH (200 mL) and HOAc (15 mL) was added sodiumcyanoborohydride (18.0 g, 286.4 mmol) in portions over a period of 30min, and the resulting solution was stirred at room temperatureovernight. The reaction was quenched by addition of saturated aqueousNaHCO₃ (500 mL) solution and the mixture was extracted with EtOAc (2×2L). The combined organic extracts were dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was purified by silica gelchromatography eluting with EtOAc/petroleum ether (10:1) to afford 40 gof Fmoc-psi-[Arg(Pbf)-Tyr(tBu)]-OAll (4) as a yellow solid.

E. Synthesis of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OAll (5)

To a solution of Fmoc-psi-[Arg(Pbf)-Tyr(tBu)-OAll] (4) (53 g, 59.28mmol) in MeCN (240 mL) was added di-tert-butyl dicarbonate (20 g, 91.3mmol), and the resulting solution was stirred at 50° C. overnight. Themixture was then concentrated under reduced pressure and the residue waspurified by silica gel chromatography eluting with EtOAc/petroleum ether(1:1) to afford 32 g of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OAll (5) as ayellow solid.

F. Synthesis of Fmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OH (6)

To a cooled (−30° C.) solution ofFmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OAll (5) (32 g, 32 mmol) in DCM (600mL) under an inert atmosphere of nitrogen was added Pd(PPh₃)₄(3.0 g,4.33 mmol), followed by drop-wise addition of N-methylaniline (10 g, 93mmol) over a period of 30 min. The resulting mixture was stirred at roomtemperature for 2 h, and then concentrated under reduced pressure. Theresidue was purified by silica gel chromatography eluting withEtOAc/petroleum ether (1:1) to afford 26.8 g ofFmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OH (6) as a yellowish solid. ¹H NMR(300 MHz, CD₃OD) δ 7.75-7.77 (2H, m), 7.59-7.60 (2H, m), 7.32-7.33 (4H,m), 7.09-7.11 (2H, m), 6.87-7.00 (2H, m), 4.27-4.50 (3H, m), 3.30-3.50(4H, m), 3.02-3.23 (3H, m), 2.75-2.98 (3H, m), 2.57 (3H, s), 2.48 (3H,s), 2.00 (3H, s), 1.31-1.41 (28H, m). LC/MS (ES, m/z): mass calcd. forC₅₂H₆₇N₅O₁₀S: 953.46, found: 954.55 [M+H]⁺.

2. Loading of the Dipeptide Fmoc-Psi-(R35-N(Boc)-Y36) onto Sieber Resin

In a fritted microwave reaction vessel (supplied by CEM Corporation),NovaSyn TG Sieber resin (supplied by Novabiochem) (0.2 mmol) was treatedwith 20% piperidine in DMF (10 mL) and heated at 50° C. for 2.5 min in aCEM microwave reactor. The reaction was drained and the resin was washedwith DMF and treated again with 20% piperidine in DMF at 50° C. for 5min in a CEM microwave reactor. After draining and washing the resinwith DMF, the deprotection treatment was repeated one more time. Theresin was then treated with a solution ofFmoc-psi-[Arg(Pbf)-(N-Boc)Tyr(tBu)]-OH obtained from above (3-5 eq.),HATU (2.75-4.8 eq.) and DIEA (6-10 eq.) in DMF (4 mL) and mixed at rtfor 6 to 24 h. The mixture was drained and the resin was washedextensively with DMF, and then capped by treatment with 20% Ac₂O in DMF(5 mL) under microwave conditions at 50° C. for 5 min. The reaction wasdrained and the resin was washed extensively with DMF and DCM.

3. Synthesis of Fmoc-βA-IKPEAPGEK(Alloc)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber Resin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resin(0.2 mmol) were performed on a CEM Liberty Blue Microwave peptidesynthesizer. Standard α-Fmoc-protected amino acids were double-coupledin 3.8-fold excess relative to the initial resin loading at 50° C. for15 min using HBTU/DIEA as the coupling agents. Fmoc-Arg(Pbf)-OH wasdouble-coupled using a two-stage protocol: 25 min at rt followed by 15min at 50° C., and Fmoc-His(Trt)-OH was double-coupled using a two-stageprotocol: 4 min at rt followed by 8 min at 50° C.

4. Synthesis of Fmoc-βA-IKPEAPGEK(NH₂)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber Resin: Alloc Deprotection

The resulting resin from above was treated with a solution ofphenylsilane (25 eq.) in deoxygenated DCM (10 mL). After stirring for -2min, a solution of the Pd(PPh₃)₄(0.5 eq.) in DCM (10 mL) was added andthe resin mixture was stirred for 30 min under argon. The reaction wasdrained and the resin was washed with deoxygenated DCM. The deprotectionwas repeated with fresh reagents, after which the reaction was drainedand the resin was washed extensively with DCM and DMF.

5. Synthesis of Fmoc-βA-IKPEAPGEK(NH-dPEG₁₂-NHFmoc)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber Resin: Coupling N-Fmoc dPEG₁₂Carboxylic Acids onto 11K

The Alloc-deprotected peptide-Sieber resin from above was treated with asolution of the N-Fmoc-dPEG12-carboxylic acid (5 eq), HBTU (4.8 eq.) andDIEA (10 eq.) in DMF (7 mL) in a CEM microwave reactor at 50° C. for 15min, by which time the reaction showed a negative Kaiser test. Thereaction was drained and the resin was washed extensively with DMF andDCM.

6. Synthesis of BrCH₂COHN-βA-IKPEAPGEK(NH-dPEG₁₂-NHCOCH₂Br)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber Resin: bis-bromoacetylation atβA and dPEG₁₂

The above resin was subjected to Fmoc-deprotection using fresh 20%piperidine in DMF at 50° C. for 5 min in a CEM microwave reactor. Thedeprotection was repeated twice. The Fmoc-deprotected peptide-resin thusobtained was treated with a solution of bromoacetic anhydride (20 eq.)in DMF (5 mL) in a CEM microwave reactor at 50° C. for 10 min, by whichtime the reaction showed a negative Kaiser test. The reaction wasdrained, and the resin was washed extensively with DMF and DCM, and thendried.

7. Synthesis of BrCH₂COHN-βA-IKPEAPGEK(NH-dPEG₁₂-NHCOCH₂Br)ASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-CONH₂: Cleavage from Resin and GlobalDeprotection

The dried resin was treated with a solution of 1.5% TFA in DCM (10 mL)and mixed for 5 to 10 min, then filtered. This treatment was repeatedfor 9 additional times using fresh cocktail for each treatment. Thecombined filtrates were then combined and concentrated to afford thecrude protected peptide as a yellow foam. This foam was treated with 20mL of cleavage cocktail (TFA/phenol/H₂O/TIPS=88/5/5/2) at roomtemperature for 2.5 h and then concentrated under a stream of nitrogento a volume of ˜2.5 mL, cold ether (40 mL) was then added to precipitatethe peptide. The mixture was centrifuged (5 min; 5000 rpm) and decanted.This process was repeated for 2 more times to give the crude peptide asan off-white powder.

Alternatively, the resin was treated with cleavage cocktail withoutprior treating with 1-2% TFA in DCM to afford the fully deprotectedpeptide.

8. Cyclic PYY Analog SEQ ID NO: 1: Cyclization Procedure a andPurification

The crude peptide from above was dissolved in deoxygenated 50%MeCN/water (5-10 mg/mL), EDTA (1 mM) was added optionally. The pH of thereaction solution was then raised to about 8 through the addition of 7.5w/v % NaHCO₃ solution. The resulting solution was stirred at rt for 0.5to 2.5 h, and then acidified to pH<1 by addition of TFA. The solutionwas then concentrated under reduced pressure at rt to about half of theoriginal volume (˜24 mL). The resultant solution was purified by reversephase preparative HPLC. Purifications were performed on a Gilson HPLC2020 Personal Purification System using a Varian Pursuit XRs C18 column(30×250 mm, 100 Å, 5 μm). The mobile phase consisted of gradient elutionof Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) rangingfrom an initial concentration of 20% B to final concentration of 50% Bover 36 min. UV detection was monitored at 220 and 254 nm.Product-containing fractions were analyzed by analytical HPLC on anAgilent 1100 HPLC system using the same column type as above (4.6×250mm, 5 μm). Pure fractions were combined, and then lyophilized to givethe product as a cotton-like solid. LCMS: 1225.5 (M+4H)/4, 1633.4(M+3H)/3 and 2450.0 (M+2H)/2 for the product peak at 12.27 min (LC:Atlantis T3 C18 column, 5 um, 4.6×250 mm, 1.0 mL/min, 15-60% gradient).

Example 2: Synthesis of Cyclic PYY Analog SEQ ID NO:2 1. Synthesis ofH₂N-IKPEAPGEDASPEELNRYYASLRHYLNL(hC) TRQRY-PAL-PEG Resin

The protected peptidyl resin was synthesized using Fmoc strategy asdescribed above on a CEM Liberty Blue Microwave peptide synthesizerusing low loading Rink amide resins, preferably, Fmoc-PAL-PEG PS resin(ca., 0.16-0.2 meq/g, supplied by Applied Biosystems) on a scale of 0.1mmol, as depicted in Scheme 1. Standard Fmoc-protected amino acids werecoupled in 5-fold excess relative to resin loading using DIC/Oxyma asthe coupling agents and a reaction temperature of ca., 90° C. for 4 min.Fmoc-Arg(Pbf)-OH was double coupled at 90° C. for 4 min each andFmoc-His(Trt)-OH was coupled using a two-stage protocol: 4 min at rtfollowed by 8 min at 50° C. Single Fmoc deprotections were carried outusing 20% piperidine in DMF (deprotection solution) at 90° C. for 1.5min.

2. Synthesis of m-BrCH₂PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG Resin

The Fmoc-deprotected peptide-resin (0.1 mmol) from above was treatedwith a solution of m-bromomethylbenzoic acid (20 eq.) and DIC (10 eq.)in DMF (4 mL) in a microwave reactor at 75° C. for 15 min, by which timethe reaction was generally determined to be complete, as per a Kaiserninhydrin test (Kaiser, et al., Anal. Biochem., 1970, 34, 595-598). Incases where the coupling was determined to be incomplete, the couplingwas repeated with fresh reagents. The reaction was drained, and theresin was washed extensively with DMF and DCM.

3. Synthesis of m-BrCH₂PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQRY-CONH₂: Deprotection and Cleavage from Resin

The resin from above was then treated with a cleavage cocktail (10mL/0.1 mmol scale) consisting of TFA/water/phenol/TIPS (88:5:5:2) andheated in a microwave reactor at 38° C. for 40 min, then filtered. Theresin was washed with TFA and the combined filtrates were concentratedunder a stream of nitrogen to a volume of ca. 2.5 mL and the peptide wasprecipitated by the addition of cold diethyl ether (40 mL). Thepeptide/ether suspension was centrifuged and the ether layer wasdecanted. The peptide pellet was re-suspended in ether, centrifuged anddecanted, and this process was repeated a third time. The crude peptidethus obtained was dried under a mild nitrogen stream.

4. Cyclic PYY Analog SEQ ID NO: 2: Cyclization Procedure a andPurification

The crude peptide from above was dissolved in deoxygenated MeCN/water(60% MeCN) at a concentration of ≤4 mg/mL. The pH of the peptidesolution was then raised to ca. 7-9 through the addition of aq. NH₄OAc(200 mM, pH 8.4) and the resulting solution was stirred at rt until thecyclization was complete, as per LCMS (typically, 3-4 h). Thecyclization reaction mixture was acidified to pH 1.5-3 by the additionof TFA, and the solution was concentrated to remove most of the organicco-solvent to a point where slight clouding occurred. A minimal amountof the MeCN was added back as necessary to render the mixturehomogeneous and the resultant solution was then purified directly bypreparative HPLC in multiple injections using a C18 Varian Pursuit XRsC18 (21×250 mm, 100 Å, 5 μm) column. The mobile phase consisted ofgradient elutions of buffer A (0.1% TFA in water) and Buffer B (0.1% TFAin MeCN) ranging from an initial concentration of 20% B to a finalconcentration of 40% B over 45 min. UV detection was monitored at 220and 254 nm. Product-containing fractions were analyzed by analyticalHPLC on an Agilent 1100 HPLC system using an appropriate column aslisted in Table 1. Pure fractions were combined, concentrated to removemost of the organic phase, and then lyophilized.

Example 3: Synthesis of Cyclic PYY Analog SEQ ID NO:3 1. Synthesis of(H₂N)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-norLeu)-TRQRYPAL-PEG Resin

The resin-bound peptide was prepared on a 0.1 mmol scale according tothe method described in Example 2, step 1, substitutingFmoc-azidonorLeu-OH in place of Fmoc-hCys(trt)-OH at position 31.

2. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-norLeu)-TRQRYPAL-PEGResin

4-Pentynoic acid was coupled onto the above resin under microwaveconditions using a DIC/HOBT protocol (75° C., 10 min). The reaction wasdrained and the resin was washed extensively with DMF and DCM.

3. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNL-(azido-norLeu)-TRQRYPAL-CONH₂

The above resin was treated with 10 mL cleavage cocktail consisting ofTFA/DODT/H₂O/TIS (92.5:2.5:2.5:2.5) under microwave conditions (38° C.,40 min). The reaction was drained and the resin was washed with TFA (10mL). The combined filtrate was then concentrated under a stream ofnitrogen to a volume of ˜2.5 mL. Cold ether (40 mL) was then added toprecipitate the peptide and the mixture was centrifuged (5 min; 5000rpm) and decanted. This process was repeated for 2 more times to givethe crude peptide as an off-white powder.

4. Cyclic PYY Analog SEQ ID NO: 3

Prepare 7 mg of CuSO₄ in 2 mL deoxygenated H₂O. Prepare 30 mg of TBTA in5.4 mL of EtOH and 0.6 mL of MeCN. Premix 0.94 mL of CuSO₄ solution and4.8 mL TBTA solution. Prepare 30 mg of Na Ascorbate in 3 mL ofdeoxygenated H₂O.

To a solution of the crude azido-containing peptide from Step 3 (100 mg)in 20 mL of deoxygenated water was added the premixed CuSO₄/TBTAsolution followed by 2.4 mL of Na ascorbate solution (solutionimmediately became milky). The mixture was warmed to 40° C. and stirredfor 1.5 h, at which time LCMS analysis indicated a complete reaction.The mixture was diluted to ˜40 mL with water (0.1% TFA); the mixture wascentrifuged, and the supernatant was purified by reverse phasepreparative HPLC. Purifications were performed using a Varian PursuitXRs C18 column (21×250 mm, 100 Å, 5 μm) at 35° C. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from an initial concentration of 10%B to an intermediate concentration of 18% B (21 mpm) and then to a finalconcentration of 33% B (10.5 mpm) over 35 min. UV detection wasmonitored at 220 and 254 nm. Product-containing fractions were analyzedby analytical HPLC on an Agilent 1100 HPLC system using the same columntype as above (4.6×250 mm, 5 μm). Pure fractions were combined, and thenlyophilized to give the product as a cotton-like solid.

Example 4: Synthesis of Cyclic PYY Analog SEQ ID NO:4 1. Synthesis of(Dde)K(NH₂)ASPEELNRYYASLRHYLNL(hC) TRQRY-PAL-PEG Resin

The resin-bound peptide was prepared using the method described inExample 2, step 1.

2. Synthesis of (Dde)K(NH-Glu-(OtBu)NH₂)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG Resin

Fmoc-Glu-OtBu (5 eq.) was coupled onto the above resin under microwaveconditions using DIC/Oxyma coupling methods (90° C., 6 min; dc). Theresin was drained and washed with DMF. Fmoc deprotection was thencarried out using 20% piperidine in DMF using a 3-stage protocol (75° C.for 0.5 min; 75° C. for 3 min; 75° C. for 3 min) with DMF washings ateach stage.

3. Synthesis of (Dde)K(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG Resin

Palmitic acid (5 eq.) was coupled onto the above resin under microwaveconditions using DIC/Oxyma coupling methods (90° C., 5 min). The resinwas drained and washed with extensively with DMF and DCM.

4. Synthesis of (H₂N)K(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC)TRQRY-PAL-PEG Resin

After washing the above resin with DMF, it was treated with a solutionof 2% hydrazine in DMF (6 mL/0.1 mmol resin) at rt for 5 min, thendrained and washed with DMF. The treatment was repeated 5 additionaltimes.

5. Synthesis of(H₂N)IKPEAPGEK(NH-Glu-(OtBu)NH-Pal)ASPEELNRYYASLRHYLNL(hC) TRQRY-PAL-PEGResin

The remaining amino acid couplings were carried out using the methoddescribed in Example 2, step 1.

6. Cyclic PYY Analog SEQ ID NO: 4

The remainder of the synthesis was carried out using the methodsdescribed in Example 2, steps 2-4. Product purification was performedusing a Varian Pursuit XRS C18 column (21×250 mm, 100 Å, 5 μm) at rt.The mobile phase consisted of a gradient elution of Buffer A (0.1% TFAin water) and Buffer B (0.1% TFA in MeCN) ranging from 23% B to anintermediate concentration of 33% B (21 mpm) over 5 min, and then to afinal concentration of 48% B (10.5 mpm) over 55 min.

Example 5: Synthesis of Cyclic PYY Analog SEQ ID NO:5

The title compound was prepared according to the procedure as describedin Example 4 substituting α-Tocopheryloxyacetic Acid (AcVitE) (8) inplace of palmitic acid in step 3. Product purification was performedusing an Agilent 300SB C8 column (21×250 mm, 100 Å, 5 μm) at rt. Themobile phase consisted of a gradient elution of Buffer A (0.1% TFA inwater) and Buffer B (0.1% TFA in MeCN) ranging from an initialconcentration of 35% B to an intermediate concentration of 45% B (21mpm) over 5 min, and then to a final concentration of 60% B (10.5 mpm)over 60 min.

Example 6: Synthesis of Cyclic PYY Analog SEQ ID NO:6 1. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(Dde)-(azido-norLeu)-TRQRY-PAL-PEGResin

The resin-bound peptide was prepared as described in Example 3, step 1,substituting Fmoc-Lys(Dde)-OH in place of Fmoc-Leu-OH at position 30 andincorporating the 4-pentynoic acid (double coupled) in this step atposition 2, following Fmoc-Ile-OH at position 3.

2. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH₂)-(azido-norLeu)-TRQRY-PAL-PEGResin

The above resin was treated with 3% hydrazine in DMF (8 mL/0.1 mmolscale) for 5 min at rt and then the mixture was drained and washed withDMF. This procedure was repeated ca. 5×, after which the resin waswashed extensively with DMF and then DCM.

3. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH-γ-Glu-AcVitE)-(azido-norLeu)-TRQRY-PAL-PEGResin

Fmoc-Glu-OtBu was coupled onto the above resin using the couplingprotocol described in Example 2, step 1 with a 5 min coupling time. Theresin was deprotected by treatment with 20% piperidine in DMF using a3-stage microwave protocol (75° C., 0.5 min; 75° C., 3 min; 75° C., 3min), after which the resin was washed extensively with DMF and DCM.α-Tocopheryloxyacetic Acid (AcVitE) (8) was then coupled onto the resinusing the same procedure used for coupling Fmoc-Glu-OtBu.

4. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH-γ-Glu-AcVitE)-(azido-norLeu)-TRQRY-CONH₂

Cleavage and precipitation of the peptide from the above resin wascarried out using the procedure described in Example 3, step 3.

5. Cyclic PYY Analog SEQ ID NO: 6

The title compound was prepared using the procedure described in Example3, step 4. Product purification was performed on a Varian Pursuit XRs C8column (21×250 mm, 100 Å, 5 μm) at 35° C. The mobile phase consisted ofa gradient elution of Buffer A (0.1% TFA in water) and Buffer B (0.1%TFA in MeCN) ranging from an initial concentration of 35% B to anintermediate concentration of 48% B (21 mpm) over 5 min, and then to afinal concentration of 63% B (10.5 mpm) over 40 min.

Example 7: Synthesis of Cyclic PYY Analog SEQ ID NO:7

The title compound was prepared according to the procedure as describedin Example 4 with the K(NH-γ-Glu-Pal) residue installed at position 9instead of position 11. Product purification was performed using anAgilent 300SB C8 column (21×250 mm, 100 Å, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from an initial concentration of 23%B to an intermediate concentration of 43% B (21 mpm) and then to a finalconcentration of 43% B (10.5 mpm) over 40 min. Impure product-containingfractions were re-purified on a Waters T3 C18 column (250×19 mm, 100 Å,5 μm) at rt using a gradient from an initial concentration of 25% B toan intermediate concentration of 35% B (21 mpm) and then to a finalconcentration of 45% B (10.5 mpm) over 80 min.

Example 8: Synthesis of Cyclic PYY Analoγ SEQ ID NO:8

The title compound was prepared according to the procedure as describedin Example 4 with the K(NH-γ-Glu-Pal) residue installed at position 30instead of position 11. Product purification was performed using anAgilent 300SB C8 column (21×250 mm, 100 Å, 5 μm) at 35° C. The mobilephase consisted of a gradient elution of Buffer A (0.1% TFA in water)and Buffer B (0.1% TFA in MeCN) ranging from an initial concentration of21% B to an intermediate concentration of 31% B (21 mpm) and then to afinal concentration of 41% B (10.5 mpm) over 40 min. Impureproduct-containing fractions were re-purified on a Waters T3 C18 column(250×19 mm, 100 Å, 5 μm) at rt using a gradient from an initialconcentration of 21% B to an intermediate concentration of 31% B (21mpm) and then to a final concentration of 40% B (10.5 mpm) over 80 min.

Example 9: Synthesis of Cyclic PYY Analog SEQ ID NO:9 1. Synthesis ofH₂N-IKPEAPGEDASPEELNRYYASLRHYLNL(hC) TRQ(psi-R35Y36)-Sieber Resin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resinfrom Example 1, step 2 (0.1 mmol) were performed as described in Example1, step 3 with the modification of using a 5-fold excess of protectedamino acids.

2. Synthesis of m-BrCH₂PhCOHN-IKPEAPGEDASPEELNRYYASLRHYLNL(hC)TRQ(psi-R35Y36)-Sieber Resin

m-Bromomethylbenzoic acid was coupled onto the above resin according tothe procedure described in Example 1, step, with the modification thatthe coupling was carried out at 50° C. instead of 75° C.

3. Cyclic PYY Analog SEQ ID NO: 9

The title compound was prepared from the above resin following theprocedures described in Example 1, steps 7 and 8. Product purificationwas performed using a Varian Pursuit XRs C18 column (21×250 mm, 100 Å, 5μm) at 35° C. The mobile phase consisted of a gradient elution of BufferA (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging from aninitial concentration of 10% B to an intermediate concentration of 18% B(21 mpm) over 10 min, and then to a final concentration of 33% B (10.5mpm) over 35 min.

Example 10: Synthesis of Cyclic PYY Analog SEQ ID NO:10

The title compound was prepared according to the procedure as describedin Example 4 substituting Dde-Lys(Fmoc)-OH in place of Fmoc-Leu-OH atposition 30 and α-Tocopheryloxyacetic Acid (AcVitE) (8) in place ofpalmitic acid in step 3. Product purification was performed using anAgilent 300SB C8 column (21×250 mm, 100 Å, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from an initial concentration of 30%B to an intermediate concentration of 40% B (21 mpm) over 10 min, andthen to a final concentration of 55% B (21 mpm) over 35 min. Impureproduct-containing fractions were re-purified using a modified gradientfrom an initial concentration of 35% B to an intermediate concentrationof 43% B (21 mpm) over 5 min, and then to a final concentration of 58% B(10.5 mpm) over 40 min.

Example 11: Synthesis of Cyclic PYY Analog SEQ ID NO:11 1. Synthesis of(Alloc)K(NH₂)-(hC)-TRQ(psi-R35Y36)-Sieber Resin

The above resin was prepared following the procedure described inExample 9, step 1 using Alloc-Lys(Fmoc)-OH in place of Fmoc-Leu-OH atposition 30.

2. Synthesis of (Alloc)K(NH-γ-Glu-AcVitE)-(hC)-TRQ(psi-R35Y36)-SieberResin

Fmoc-Glu-OtBu and α-Tocopheryloxyacetic Acid (AcVitE) (8) (5 eq. each)were sequentially coupled onto the above resin using HBTU/DIEA-mediatedcouplings under microwave conditions at 50° C. for 15-20 min.

3. Synthesis of H₂N-K(NH-γ-Glu-AcVitE)-(hC)-TRQ(psi-R35Y36)-Sieber Resin

The alloc protecting group was removed following the procedure describedin Example 1, step 4.

4. Cyclic PYY Analog SEQ ID NO: 11

The title compound was prepared from the above resin following theprocedures described in Example 9, steps 1-3, with the modification thata 1M TRIS/HCl buffer, pH 7.5 was used in place of the NH₄OAc buffer toeffect cyclization. Product purification was performed using a VarianPursuit XRs C18 column (21×250 mm, 100 Å, 5 μm) at 35° C. The mobilephase consisted of a gradient elution of Buffer A (0.1% TFA in water)and Buffer B (0.1% TFA in MeCN) ranging from an initial concentration of10% B to an intermediate concentration of 18% B (21 mpm) over 10 min,and then to a final concentration of 33% B (10.5 mpm) over 35 min.

Example 12: Synthesis of Cyclic PYY Analog SEQ ID NO:12

The title compound was prepared according to the procedure as describedin Example 2 substituting Fmoc-N-Me-Arg(pbf)-OH in place ofFmoc-Arg(pbf)-OH at position 35 in step 1 and with the modification thata 1M NaHCO₃ buffer was used in place of the NH₄OAc buffer to effectcyclization. Product purification was performed using a Varian PursuitXRs C18 column (30×250 mm, 100 Å, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from 10-60% B (30 mpm) over 36 min.

Example 13: Synthesis of Cyclic PYY Analog SEQ ID NO:13

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8) in step 2, and with the modifications that 60% EtOH/H₂O wasused as solvent in place of MeCN/H₂O and sat'd aq. NaHCO₃ was used inplace of the NH₄OAc buffer to effect cyclization. Product purificationwas performed using a Waters XBridge C18 OBD column (50×250 mm, 5 μm) atrt. The mobile phase consisted of a gradient elution of Buffer A (10 mMNH₄OH in water, pH˜9) and Buffer B (MeCN) ranging from an initialconcentration of 15% B to an intermediate concentration of 20% B (100mpm) over 5 min, and then to a final concentration of 35% B (100 mpm)over 40 min.

Example 14: Synthesis of Cyclic PYY Analog SEQ ID NO:14

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11 and with Fmoc-N-Me-Arg(pbf)-OH in place ofFmoc-Arg(pbf)-OH at position 35 in step 1 and with the modification thata 1M NaHCO₃ buffer was used in place of the NH₄OAc buffer in step 6, toeffect cyclization. Product purification was performed using a VarianPursuit XRs C18 column (30×250 mm, 100 Å, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from 20-70% B (30 mpm) over 36 min.Impure fractions were re-purified on a Varian Pursuit XRs diphenylcolumn (30×100 mm, 100 Å, 5 μm) at rt using a gradient of 30-50% B (30mpm) over 25 min.

Example 15: Synthesis of Cyclic PYY Analog SEQ ID NO:15

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, substituting Fmoc-βAla-OH in place ofFmoc-Ile-OH coupling at position 3, and with the modifications that a 1MNaHCO₃ buffer was used in place of the NH₄OAc buffer to effectcyclization, and coupling with bromoacetic anhydride was used in placeof m-bromomethylbenzoic acid in step 6 (step 2 from Example 2) using thefollowing procedure: The Fmoc-deprotected peptide-resin (0.1 mmol) wastreated with a solution of bromoacetic anhydride (10 eq.) in DMF (5 mL)in a microwave reactor at 50° C. for 5-10 min, by which time thereaction was generally determined to be complete as per a Kaiserninhydrin test. In cases where the coupling was determined to beincomplete, the coupling was repeated with fresh reagents. Productpurification was performed using a Varian Pursuit XRs C18 column (30×250mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradient elutionof Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) rangingfrom 20-60% B (30 mpm) over 36 min. Impure fractions were re-purified ona Varian Pursuit XRs diphenyl column (30×100 mm, 100 Å, 5 μm) at rtusing a gradient of 30-50% B (30 mpm) over 25 min.

Example 16: Synthesis of Cyclic PYY Analog SEQ ID NO:16

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, omitting the Fmoc-Ile-OH coupling at position 3,and using p-bromomethylbenzoic acid in place of m-bromomethylbenzoicacid in step 6 (step 2 from Example 2). Additionally, a 1M NaHCO₃ bufferwas used in place of the NH₄OAc buffer, to effect cyclization. Productpurification was performed using a Varian Pursuit XRs C18 column (30×250mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradient elutionof Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) rangingfrom 20-70% B (30 mpm) over 36 min. Impure fractions were re-purified ona Varian Pursuit XRs diphenyl column (30×100 mm, 100 Å, 5 μm) at rtusing a gradient of 30-50% B (30 mpm) over 25 min.

Example 17: Synthesis of Cyclic PYY Analog SEQ ID NO:17

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11 and Fmoc-Ala-OH used in place of Fmoc-Lys(Boc)-OHat position 4 in step 1. TRIS/HCl buffer (1M, pH 7.5) was used in placeof the NH₄OAc buffer in step 6 to effect cyclization. Productpurification was performed using an Agilent Polaris C18-A column (30×250mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradient elutionof Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) rangingfrom an initial concentration of 20% B to an intermediate concentrationof 35% B (40 mpm) over 5 min, and then to a final concentration of 45% B(40 mpm) over 40 min.

Example 18: Synthesis of Cyclic PYY Analog SEQ ID NO:18

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11 and Fmoc-Glu(OtBu)-OH used in place ofFmoc-Lys(Boc)-OH at position 4 in step 1. TRIS/HCl buffer (1M, pH 7.5)was used in place of the NH₄OAc buffer in step 6 to effect cyclization.Product purification was performed using an Agilent Polaris C18-A column(30×250 mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradientelution of Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN)ranging from an initial concentration of 20% B to an intermediateconcentration of 35% B (40 mpm) over 5 min, and then to a finalconcentration of 45% B (40 mpm) over 40 min.

Example 19: Synthesis of Cyclic PYY Analog SEQ ID NO:19

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, Fmoc-N-Me-Arg(pbf)-OH in place ofFmoc-Arg(pbf)-OH at position 35 in step 1, Fmoc-Cys(trt)-OH in place ofFmoc-hCys(trt)-OH, and using p-bromomethylbenzoic acid in place ofm-bromomethylbenzoic acid in step 6 (step 2 from Example 2).

The following modification was made to step 6 (steps 3 and 4 fromExample 2): The crude peptide obtained prior to cyclization was purifiedusing a Varian Pursuit XRs C18 column (30×250 mm, 100 Å, 5 μm) at rt.The mobile phase consisted of a gradient elution of Buffer A (0.1% TFAin water) and Buffer B (0.1% TFA in MeCN) ranging from 20-70% B (30 mpm)over 36 min. Product-containing fractions were combined and treated withsolid NaHCO₃ to raise the pH to ˜7-8; the resulting solution was stirredat rt for 4 h, then acidified to pH 4 with TFA. The solution wasconcentrated to a volume of 5-10 mL and MeCN was added to solubilize anyprecipitate. Product purification was performed as above, with agradient of 20-60% B (30 mpm) over 36 min.

Example 20: Synthesis of Cyclic PYY Analog SEQ ID NO:20

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, Fmoc-N-Me-Arg(pbf)-OH in place ofFmoc-Arg(pbf)-OH at position 35 in step 1 and Fmoc-Cys(trt)-OH in placeof Fmoc-hCys(trt)-OH. The crude linear peptide was purified and cyclizedaccording to the modification described in Example 19. Final productpurification was performed using a gradient of 20-60% B (30 mpm) over 36min.

Example 21: Synthesis of Cyclic PYY Analog SEQ ID NO:21

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, Fmoc-Ala-OH used in place of Fmoc-His(trt)-OH atposition 26 and Fmoc-Ala-OH used in place of Fmoc-Lys(Boc)-OH atposition 4 in step 1. TRIS/HCl buffer, (1M, pH 7.5) was used in place ofthe NH₄OAc buffer in step 6 to effect cyclization. Product purificationwas performed using an Agilent Polaris C18-A column (30×250 mm, 100 Å, 5μm) at rt. The mobile phase consisted of a gradient elution of Buffer A(0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging from aninitial concentration of 20% B to an intermediate concentration of 35% B(40 mpm) over 5 min, and then to a final concentration of 45% B (40 mpm)over 40 min.

Example 22: Synthesis of Cyclic PYY Analog SEQ ID NO:22

The title compound was prepared according to the procedure as describedin Example 4 with the Lys(NH-γ-Glu-Pal) residue installed at position 30instead of position 11, Fmoc-Ala-OH used in place of Fmoc-His(trt)-OH atposition 26 and Fmoc-Glu(OtBu)-OH used in place of Fmoc-Lys(Boc)-OH atposition 4 in step 1. TRIS/HCl buffer, (1M, pH 7.5) was used in place ofthe NH₄OAc buffer in step 6 to effect cyclization. Product purificationwas performed using an Agilent Polaris C18-A column (30×250 mm, 100 Å, 5μm) at rt. The mobile phase consisted of a gradient elution of Buffer A(0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging from aninitial concentration of 20% B to an intermediate concentration of 33% B(40 mpm) over 5 min, and then to a final concentration of 43% B (40 mpm)over 40 min.

Example 23: Synthesis of Cyclic PYY Analog SEQ ID NO:23

The title compound was prepared according to the procedures described inExample 11, using octadecanedioic acid, mono-tert-butyl ester (availablefrom AstaTech, Inc.) in place of α-Tocopheryloxyacetic Acid (AcVitE)(8), a coupling protocol employing HATU/DIEA at 50° C. for 30 min andNMP as solvent in place of DMF in step 2, and coupling two units ofFmoc-OEG-OH in tandem prior to coupling Fmoc-Glu-OtBu, in step 2. Thecrude linear peptide was purified and cyclized according to themodification described in Example 19, using a gradient of 20-60% B.Final product purification was performed using a gradient of 20-70% B(30 mpm) over 36 min.

Example 24: Synthesis of Cyclic PYY Analog SEQ ID NO:24

The title compound was prepared according to the procedures described inExample 23, using 20-(tert-butoxy)-20-oxoicosanoic acid (available fromKey Organics, Inc.) in place of octadecanedioic acid, mono-tert-butylester. The crude linear peptide was purified and cyclized according tothe modification described in Example 19, using a gradient of 20-60% B.Final product purification was performed using a gradient of 20-60% B(30 mpm) over 36 min.

Example 25: Synthesis of Cyclic PYY Analog SEQ ID NO:25

The title compound was prepared according to the procedures described inExample 11, using stearic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), a coupling protocol employing HATU/DIEA at 50° C. for 30min and NMP as solvent in place of DMF in step 2. The crude linearpeptide was purified and cyclized according to the modificationdescribed in Example 19, using a gradient of 20-80% B. Final productpurification was performed using a gradient of 20-80% B (30 mpm) over 36min.

Example 26: Synthesis of Cyclic PYY Analog SEQ ID NO:26

The title compound was prepared according to the procedures described inExample 11, using arachidic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), a coupling protocol employing HATU/DIEA at 50° C. for 30min and NMP as solvent in place of DMF in step 2. The crude linearpeptide was purified and cyclized according to the modificationdescribed in Example 19, using a gradient of 20-90% B. Final productpurification was performed using a gradient of 20-90% B (30 mpm) over 36min.

Example 27: Synthesis of Cyclic PYY Analog SEQ ID NO:27

The title compound was prepared according to the procedures described inExample 23, but omitting the coupling of Fmoc-Glu-OtBu after the tandemFmoc-OEG-OH couplings. The crude linear peptide was purified andcyclized according to the modification described in Example 19, using agradient of 20-60% B. Final product purification was performed using agradient of 20-60% B (30 mpm) over 36 min.

Example 28: Synthesis of Cyclic PYY Analog SEQ ID NO:28

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), omitting the coupling of Fmoc-Ile-OH at position 3, andusing p-bromomethylbenzoic acid in place of m-bromomethylbenzoic acid instep 4 (Example 9, step 2). The crude linear peptide was purified andcyclized according to the modification described in Example 19, using agradient of 20-60% B. Final product purification was performed using agradient of 20-60% B (30 mpm) over 36 min.

Example 29: Synthesis of Cyclic PYY Analog SEQ ID NO:29

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), substituting Fmoc-βAla-OH in place of Fmoc-Ile-OH atposition 3, and coupling bromoacetic anhydride in place ofm-bromomethylbenzoic acid in step 4 (Example 9, step 2), using themodification described in Example 15. The crude linear peptide waspurified and cyclized according to the modification described in Example19, using a gradient of 20-60% B. Final product purification wasperformed using a gradient of 20-60% B (30 mpm) over 36 min.

Example 30: Synthesis of Cyclic PYY Analog SEQ ID NO:30

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), substituting Fmoc-Aβυ-OH in place of Fmoc-Ile-OH atposition 3, and coupling bromoacetic anhydride in place ofm-bromomethylbenzoic acid in step 4 (Example 9, step 2), using themodification described in Example 15. The crude linear peptide waspurified and cyclized according to the modification described in Example19, using a gradient of 20-60% B. Final product purification wasperformed using a gradient of 20-60% B (30 mpm) over 36 min.

Example 31: Synthesis of Cyclic PYY Analog SEQ ID NO:31

The title compound was prepared according to the procedures described inExample 23, using stearic acid in place of octadecanedioic acid,mono-tert-butyl ester. The crude linear peptide was purified andcyclized according to the modification described in Example 19, using agradient of 20-60% B. Final product purification was performed using agradient of 20-60% B (30 mpm) over 36 min.

Example 32: Synthesis of Cyclic PYY Analog SEQ ID NO:32

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8) in step 2, Fmoc-Ala-OH used in place of Fmoc-His(trt)-OH atposition 26 and Fmoc-Ala-OH used in place of Fmoc-Lys(Boc)-OH atposition 4 in step 4 (Example 9, step 1). TRIS/HCl buffer, (1M, pH 7.5)was used in place of the NH₄OAc buffer in step 6 to effect cyclization.Product purification was performed using an Agilent Polaris C18-A column(30×250 mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradientelution of Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN)ranging from an initial concentration of 20% B to an intermediateconcentration of 35% B (40 mpm) over 5 min, and then to a finalconcentration of 45% B (40 mpm) over 40 min.

Example 33: Synthesis of Cyclic PYY Analog SEQ ID NO:33

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8) in step 2 and Fmoc-N(Me)-Gln(trt)-OH in place ofFmoc-Gln(trt)-OH at position 34 in step 4 (Example 9, step 1). In thiscase, couplings were carried out at rt using NMP as solvent and anHATU/DIEA protocol (1 h, single coupling); Fmoc-N(Me)-Gln(trt)-OH andFmoc-Arg(pbf)-OH were double coupled. A two-stage Fmoc deprotectionprotocol was used throughout (20% piperidine in DMF; rt; 10 min, 15min). The crude linear peptide was purified and cyclized according tothe modification described in Example 19, using a gradient of 20-70% B.Final product purification was performed using a gradient of 20-70% B(30 mpm) over 36 min.

Example 34: Synthesis of Cyclic PYY Analog SEQ ID NO:34

The title compound was prepared according to the procedures described inExample 11, using palmitic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8), substituting Fmoc-Cys(trt)-OH in place ofFmoc-hCys(trt)-OH at position 31,6-Fmoc-aminohexanoic acid in place ofFmoc-Ile-OH at position 3, and coupling bromoacetic anhydride in placeof m-bromomethylbenzoic acid in step 4 (Example 9, step 2), using themodification described in Example 15. Aq. NaHCO₃ (2N) was used in placeof the NH₄OAc buffer to effect cyclization. Product purification wasperformed using a Varian Pursuit XRs C18 column (30×250 mm, 100 Å, 5 μm)at rt. The mobile phase consisted of a gradient elution of Buffer A(0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging from 20-70%B (30 mpm) over 36 min.

Example 35: Synthesis of Cyclic PYY Analog SEQ ID NO:35

The title compound was prepared according to the procedures described inExample 9, with the following modifications:Fmoc-psi-[N-Me-Arg(Pbf)-N(Boc)Tyr(tBu)]-OH, prepared fromFmoc-N-Me-Arg(pbf)-OH in place of Fmoc-Arg(Pbf)-OH, according to theprocedure described in Example 1, step 1, was used in place ofFmoc-psi-[Arg(Pbf)-N(Boc)Tyr(tBu)]-OH (6) to prepare the loaded Sieberresin used herein; Fmoc-Lys(Pal-Glu-OtBu)-OH (from Active Peptide) wasused in place of Leu at position 30; m-chloromethylbenzoic acid was usedin place of m-bromomethylbenzoic acid in step 2; couplings were carriedout at rt using NMP as solvent and an HATU/DIEA protocol (1 h, singlecoupling) was used; Fmoc-Arg(pbf)-OH was double coupled. A two-stageFmoc deprotection protocol was used throughout (20% piperidine in DMF;rt; 10 min, 15 min). The crude linear peptide was purified and cyclizedaccording to the modification described in Example 19, using a gradientof 20-70% B. Final product purification was performed using a gradientof 20-70% B (30 mpm) over 36 min.

Example 36: Synthesis of Cyclic PYY Analog SEQ ID NO:36

The title compound was prepared according to the procedures described inExample 35, substituting Fmoc-βAla-OH in place of Fmoc-Ile-OH atposition 3, and coupling bromoacetic anhydride in place ofm-bromomethylbenzoic acid in step 4 (Example 9, step 2), using themodification described in Example 15. The modified workup of Example 19was omitted. Fmoc-βAla-OH was coupled under microwave conditions at 50°C. for 20 min. Sat'd aq. NaHCO₃ was used in place of the NH₄OAc bufferto effect cyclization. Product purification was performed using a VarianPursuit XRs C18 column (30×250 mm, 100 Å, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (0.1% TFA in water) andBuffer B (0.1% TFA in MeCN) ranging from 20-70% B (30 mpm) over 36 min.

Example 37: Synthesis of Cyclic PYY Analog SEQ ID NO:37

The title compound was prepared according to the procedures described inExample 9, substituting Fmoc-Cys(trt)-OH in place of Fmoc-hCys(trt)-OHat position 31 and Fmoc-Lys(Pal-Glu-OtBu)-OH (from Active Peptide) inplace of Fmoc-Leu-OH at position 30. In addition, Fmoc-Abu-OH wasappended onto the sequence at position 2, in step 1, and coupling withbromoacetic anhydride was used in place of m-bromomethylbenzoic acid instep 2, using the modification described in Example 15. Sat'd aq. NaHCO₃was used in place of the NH₄OAc buffer in step 3 to effect cyclization.Product purification was performed using a Varian Pursuit XRs C18 column(30×250 mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradientelution of Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN)ranging from 20-70% B (30 mpm) over 36 min.

Example 38: Synthesis of Cyclic PYY Analog SEQ ID NO:38

The title compound was prepared according to the procedures described inExample 35, with the following modifications: Fmoc-βAla-OH was appendedonto the sequence at position 2, following step 1 using microwaveconditions at 50° C. for 20 min, and coupling with bromoacetic anhydridewas used in place of m-bromomethylbenzoic acid in step 2, using themodification described in Example 15. Sat'd aq. NaHCO₃ was used in placeof the NH₄OAc buffer to effect cyclization. Product purification wasperformed using a Varian Pursuit XRs C18 column (30×250 mm, 100 Å, 5 μm)at rt. The mobile phase consisted of a gradient elution of Buffer A(0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging from 20-80%B (30 mpm) over 36 min.

Example 39: Synthesis of Cyclic PYY Analog SEQ ID NO:39

The title compound was prepared according to the procedures described inExample 11, using arachidic acid in place of α-Tocopheryloxyacetic Acid(AcVitE) (8) in step 2. Fmoc-Ser(tBu)-OH was used in place ofFmoc-Lys(Boc)-OH at position 4 in step 4 (Example 9, step 1) andm-chloromethylbenzoic acid was used in place of m-bromomethylbenzoicacid in step 4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent inplace of MeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAcbuffer in step 4 to effect cyclization. Product purification wasperformed using a Waters XBridge C18 OBD column (50×250 mm, 5 μm) at rt.The mobile phase consisted of a gradient elution of Buffer A (10 mMNH₄OH in water, pH 9) and Buffer B (MeCN) ranging from an initialconcentration of 20% B to an intermediate concentration of 25% B (100mpm) over 5 min, and then to a final concentration of 40% B (100 mpm)over 40 min.

Example 40: Synthesis of Cyclic PYY Analog SEQ ID NO:40 1. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(Dde)-(azido-norLeu)-TRQ(psi-R35Y36)-SieberResin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resinfrom Example 1, step 2 (0.1 mmol) were carried out at rt using NMP assolvent, a 5-fold excess of protected amino acids and an HATU/DIEAprotocol (1 h, single coupling); Fmoc-Arg(pbf)-OH and Fmoc-His(trt)-OHwere double coupled. A two-stage Fmoc deprotection protocol was usedthroughout (20% piperidine in DMF; rt; 10 min, 15 min).

2. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK(NH₂)-(azido-norLeu)-TRQ(psi-R35Y36)-SieberResin

The above resin was treated with 2% hydrazine in DMF (12 mL/0.2 mmolscale) for 2 min at rt and then the mixture was drained. This procedurewas repeated ca. 4×, after which the resin was washed extensively withDMF and then DCM.

3. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK((OEG)₂-γ-Glu-Pal)-(azido-norLeu)-TRQ(psi-R35Y36)-SieberResin

The above resin was coupled with(S)-10,19-dioxo-22-palmitamido-3,6,12,15-tetraoxa-9,18-diazatricosanedioicacid (5 eq,) [prepared according to the procedure described forsynthesis of intermediate 3, by substituting palmitic acid in place of18-tert-butoxy-18-oxooctadecanoic acid in step G], using an HBTU/DIEAprotocol at rt for 1.5 h. The resin was drained and washed extensivelywith DMF and DCM.

4. Synthesis of(HCCH(CH₂)₂CONH)-IKPEAPGEDASPEELNRYYASLRHYLNK((OEG)₂-γ-Glu-Pal)-(azido-norLeu)-TRQ(psi-R35Y36)-CONH₂

The dried resin was treated with a solution of 2% TFA in DCM (20 mL) andmixed for 20 min, then filtered. This treatment was repeated for 2additional times using fresh cocktail for each treatment. The combinedfiltrates were then combined and concentrated to afford the crudeprotected peptide as a yellow foam. This foam was treated with 20 mL ofcleavage cocktail (TFA/H₂O/TIPS=95/2.5/2.5) at rt for 2.5 h and thenconcentrated under a stream of nitrogen to a volume of about 2.5 mL.Cold ether (40 mL) was then added to precipitate the peptide and themixture was centrifuged (5 min; 5000 rpm) and decanted. This process wasrepeated for 2 more times to give the crude peptide as an off-whitepowder.

Alternatively, the resin was treated with cleavage cocktail withoutprior treatment with 1-2% TFA in DCM, to afford the fully deprotectedpeptide directly.

The crude peptide was purified by reverse phase preparative HPLC using aVarian Pursuit XRs C18 column (30×250 mm, 100 Å, 5 μm). The mobile phaseconsisted of gradient elution of Buffer A (0.1% TFA in water) and BufferB (0.1% TFA in MeCN) ranging from 20-70% B over 36 min. UV detection wasmonitored at 220 and 254 nm. Product-containing fractions were analyzedby analytical HPLC on an Agilent 1100 HPLC system using the same columntype as above (4.6×250 mm, 5 μm). Pure fractions were combined, and thenlyophilized to give the product as a cotton-like solid. LCMS: 1211.8(M+4H)/4, 1615.4 (M+3H)/3 and 2422.9 (M+2H)/2 for the product peak at16.87 min (LC: Atlantis T3 C18 column, 5 μm, 4.6×250 mm, 1.0 mL/min,30-60% gradient).

5. Cyclic PYY Analog SEQ ID NO: 40

Prepare 5.1 mg of CuSO₄ in 1 mL H₂O. Prepare 10.4 mg of TBTA in 3 mL ofEtOH. Premix 400 μL of CuSO₄ solution and 3 mL TBTA solution. Prepare 13mg of Na Ascorbate in 2 mL of H₂O.

To a solution of the purified azido-containing peptide from Step 4 (37mg) in 4 mL of HEPES (0.1M, pH 7.4) was added 1.7 mL of the premixedCuSO₄/TBTA solution followed by 1 mL of Na Ascorbate solution. AdjustEtOH/H₂O ratio until the reaction solution turned clear. The mixture wasstirred at rt and monitored by HPLC. After 30 min, the reaction wascompleted. The mixture was adjusted to pH 4 and purified by reversephase preparative HPLC. Purifications were performed using a VarianPursuit XRs C18 column (30×250 mm, 100 Å, 5 μm). The mobile phaseconsisted of gradient elution of Buffer A (0.1% TFA in water) and BufferB (0.1% TFA in MeCN) ranging from 20-60% B over 36 min. UV detection wasmonitored at 220 and 254 nm. Product-containing fractions were analyzedby analytical HPLC on an Agilent 1100 HPLC system using the same columntype as above (4.6×250 mm, 5 μm). Pure fractions were combined, and thenlyophilized to give the product as a cotton-like solid.

Example 41: Synthesis of Cyclic PYY Analog SEQ ID NO:41

The title compound was prepared according to the procedure described inExample 40, substituting L-Glutamic acid, N-(1-oxohexadecyl)-,1-(1,1-dimethylethyl) ester in place of(S)-10,19-dioxo-22-palmitamido-3,6,12,15-tetraoxa-9,18-diazatricosanedioicacid, in step 3.

Example 42: Synthesis of Cyclic PYY Analog SEQ ID NO:42

The title compound was prepared according to the procedure described inExample 40, substituting(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicacid (16) (intermediate 2) in place of(S)-10,19-dioxo-22-palmitamido-3,6,12,15-tetraoxa-9,18-diazatricosanedioicacid, in step 3. Example 43 Synthesis of Cyclic PYY Analog SEQ ID NO:43

1. Synthesis of (Alloc)Lys((OEG)₂-γ-Glu-NH₂)-(hC)-TRQ(psi-R35Y36)-SieberResin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resinfrom Example 1, step 2 (0.1 mmol) were carried out at rt using NMP assolvent, a 5-fold excess of protected amino acids and an HATU/DIEAprotocol (1 h, single coupling); Fmoc-Arg(pbf)-OH was double coupled. Atwo-stage Fmoc deprotection protocol was used throughout (20% piperidinein DMF; rt; 10 min, 15 min).

2. Synthesis of (Alloc)Lys((OEG)₂-γ-Glu-Pal)-(hC)-TRQ(psi-R35Y36)-SieberResin

Palmitic acid was coupled onto the resin from step 1, using microwaveconditions employing HATU/DIEA at 50° C. for 20-30 min and NMP assolvent.

3. Synthesis of (H₂N)Lys((OEG)₂-γ-Glu-Pal)-(hC)-TRQ(psi-R35Y36)-SieberResin

The alloc protecting group of the above resin was removed following theprocedure described in Example 1, step 4.

4. Cyclic PYY Analog SEQ ID NO: 43

The title compound was prepared from the above resin following theprocedures described in Example 9, steps 1-3, usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step2. The crude linear peptide was purified and cyclized according to themodification described in Example 19, using a gradient of 20-60% B.Final product purification was performed using a gradient of 20-60% B(30 mpm) over 36 min.

Example 44 Synthesis of Cyclic PYY Analog SEQ ID NO:44

The title compound was prepared according to the procedures described inExample 43, modified such that the tandem Fmoc-OEG-OH units and theFmoc-Glu-OtBu unit were incorporated in step 2 instead of step 1.Octadecanedioic acid, mono-tert-butyl ester (AstaTech, Inc.) was used inplace of palmitic acid in step 2 and the linker-lipid sequence wasinstalled at position 11 instead of position 30. The crude linearpeptide was purified and cyclized according to the modificationdescribed in Example 19, using a gradient of 20-70% B. Final productpurification was performed using a gradient of 20-70% B (30 mpm) over 36min.

Example 45 Synthesis of Cyclic PYY Analog SEQ ID NO:45

The title compound was prepared according to the procedures described inExample 43, modified such that Fmoc-dPEG24-carboxylic acid was used inplace of the tandem Fmoc-OEG-OH units and were, along with palmiticacid, incorporated into step 2. The linker-lipid sequence was installedat position 11 instead of position 30. The crude linear peptide waspurified and cyclized according to the modification described in Example19, using a gradient of 20-90% B. Final product purification wasperformed using a gradient of 20-90% B (30 mpm) over 36 min.

Example 46 Synthesis of Cyclic PYY Analog SEQ ID NO:46

The title compound was prepared according to the procedures described inExample 9, using Fmoc-Lys(Pal-Glu-OtBu)-OH (from Active Peptide) inplace of Leu at position 30. In addition, Fmoc-βAla-OH was appended ontothe sequence at position 2, following step 1 using microwave conditionsat 50° C. for 20 min, and coupling with bromoacetic anhydride was usedin place of m-bromomethylbenzoic acid in step 2, using the modificationdescribed in Example 15. Solid The crude linear peptide was purified andcyclized according to the modification described in Example 19, using agradient of 20-70% B. Final product purification was performed using agradient of 20-70% B (30 mpm) over 36 min.

Example 47 Synthesis of Cyclic PYY Analog SEQ ID NO:47

The title compound was prepared according to the procedures described inExample 44, installing the linker-lipid sequence at position 7 insteadof position 11. Product purification was performed using a VarianPursuit XRs C18 column (30×250 mm, 100 Å, 5 μm) at rt. The crude linearpeptide was purified and cyclized according to the modificationdescribed in Example 19, using a gradient of 20-60% B. Final productpurification was performed using a gradient of 20-60% B (30 mpm) over 36min.

Example 48 Synthesis of Cyclic PYY Analog SEQ ID NO:48

The title compound was prepared according to the procedures described inExample 43, using octadecanedioic acid, mono-tert-butyl ester (AstaTech,Inc.) in place of palmitic acid in step 2 with a coupling time of 30min, and installing the linker-lipid sequence at position 22 instead ofposition 30. The crude linear peptide was purified and cyclizedaccording to the modification described in Example 19, using a gradientof 20-70% B. Final product purification was performed using a gradientof 20-70% B (30 mpm) over 36 min.

Example 49 Synthesis of Cyclic PYY Analog SEQ ID NO:49

The title compound was prepared following the procedures described inExample 11, substituting 16-tetrahydropyran-2-yloxypalmitic acid inplace of α-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH˜9)and Buffer B (MeCN) ranging from an initial concentration of 20% B to anintermediate concentration of 10% B (100 mpm) over 5 min, and then to afinal concentration of 30% B (100 mpm) over 40 min.

Example 50 Synthesis of Cyclic PYY Analog SEQ ID NO:50

The title compound was prepared according to the procedures described inExample 48, and installing the linker-lipid sequence at position 23instead of position 30.

Example 51 Synthesis of Cyclic PYY Analog SEQ ID NO:51

The title compound was prepared according to the procedures described inExample 9, using Fmoc-Lys(Pal-Glu-OtBu)-OH (from Active Peptide) inplace of Fmoc-Leu-OH at position 30 and Fmoc-Ser(tBu)-OH in place ofFmoc-Lys(Boc)-OH at position 4, in step 1. 60% EtOH/H₂O was used assolvent in place of MeCN/H₂O and sat'd aq. NaHCO₃ was used in place ofthe NH₄OAc buffer in step 4 to effect cyclization. Product purificationwas performed using a Waters XBridge C18 OBD column (50×250 mm, 5 μm) atrt. The mobile phase consisted of a gradient elution of Buffer A (10 mMNH₄OH in water, pH˜9) and Buffer B (MeCN) ranging from an initialconcentration of 10% B to an intermediate concentration of 20% B (100mpm) over 5 min, and then to a final concentration of 30% B (100 mpm)over 40 min. Impure fractions were re-chromatographed using a gradientcomprised of an initial concentration of 10% B to an intermediateconcentration of 20% B (100 mpm) over 5 min, and then to a finalconcentration of 30% B (100 mpm) over 60 min.

Example 52 Synthesis of Cyclic PYY Analog SEQ ID NO:52

The title compound was prepared according to the procedures described inExample 43, using Fmoc-dPEG12-carboxylic acid in place of the tandemFmoc-OEG-OH units and incorporating it along with Fmoc-Glu-OtBu andpalmitic acid in step 2. The linker-lipid sequence was installed atposition 11 instead of position 30. The crude linear peptide waspurified and cyclized according to the modification described in Example19, using a gradient of 20-70% B. Final product purification wasperformed using a gradient of 20-70% B (30 mpm) over 36 min.

Example 53 Synthesis of Cyclic PYY Analog SEQ ID NO:53

The title compound was prepared according to the procedures described inExample 52, using four units of Fmoc-OEG-OH in tandem in place ofFmoc-dPEG12-carboxylic acid.

Example 54 Synthesis of Cyclic PYY Analog SEQ ID NO:54

The title compound was prepared according to the procedures described inExample 53, installing two units of Fmoc-OEG-OH in tandem instead oftwo.

Example 55 Synthesis of Cyclic PYY Analog SEQ ID NO:55

The title compound was prepared according to the procedures described inExample 43, installing the linker-lipid sequence at position 23 insteadof position 30. The crude linear peptide was purified and cyclizedaccording to the modification described in Example 19, using a gradientof 20-70% B. Final product purification was performed using a gradientof 20-70% B (30 mpm) over 36 min

Example 56: Synthesis of Cyclic PYY Analog SEQ ID NO:56

The title compound was prepared using the methods described in Example11, substituting (4′-chlorobiphenyl-4-yl)-acetic acid in place ofα-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH˜9)and Buffer B (MeCN) ranging from 10-28% B (100 mpm) over 40 min.

Example 57: Synthesis of Cyclic PYY Analog SEQ ID NO:57

The title compound was prepared following the procedures described inExample 11, substituting 3-[(2,4-dichlorophenoxy)phen-4-yl]propionicacid in place of α-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, andusing m-chloromethylbenzoic acid in place of m-bromomethylbenzoic acidin step 4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in placeof MeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc bufferin step 4 to effect cyclization. Product purification was performedusing a Waters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. Themobile phase consisted of a gradient elution of Buffer A (10 mM NH₄OH inwater, pH˜9) and Buffer B (MeCN) ranging from 10-30% B (80 mpm) over 40min. Product-containing fractions were combined, acidified with TFA,concentrated and re-chromatographed on an Agilent Polaris C18-A column(30×250 mm, 100 Å, 5 μm) at rt. The mobile phase consisted of a gradientelution of Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN)ranging from an initial concentration of 20% B to an intermediateconcentration of 15% B (40 mpm) to a final concentration of 45% B (40mpm) over 45 min.

Example 58: Synthesis of Cyclic PYY Analog SEQ ID NO:58

The title compound was prepared following the procedures described inExample 11, substituting 11-(4-fluorophenyl}undecanoic acid in place ofα-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH˜9)and Buffer B (MeCN) ranging from 15-35% B (100 mpm) over 40 min.

Example 59: Synthesis of Cyclic PYY Analog SEQ ID NO:59

The title compound was prepared according to the procedures described inExample 43, omitting step 2 and incorporating the palmitic acid couplinginto step 1. The linker-lipid sequence was installed at position 22instead of position 11. The crude linear peptide was purified andcyclized according to the modification described in Example 19, using agradient of 20-70% B. Final product purification was performed using agradient of 20-70% B (30 mpm) over 36 min

Example 60: Synthesis of Cyclic PYY Analog SEQ ID NO:60

The title compound was prepared according to the procedures described inExample 53, incorporating two FMOC-OEG-OH units in tandem instead offour and installing the linker-lipid sequence was at position 7 insteadof position 11. The crude linear peptide was purified and cyclizedaccording to the modification described in Example 19, using a gradientof 20-80% B. Final product purification was performed using a gradientof 20-80% B (30 mpm) over 36 min.

Example 61: Synthesis of Cyclic PYY Analog SEQ ID NO:61

The title compound was prepared following the procedures described inExample 11, substituting 11-[(4-trifluoromethyl)phenyl]undecanoic acidin place of ca-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, andusing m-chloromethylbenzoic acid in place of m-bromomethylbenzoic acidin step 4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in placeof MeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc bufferin step 4 to effect cyclization. Product purification was performedusing a Waters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. Themobile phase consisted of a gradient elution of Buffer A (10 mM NH₄OH inwater, pH˜9) and Buffer B (MeCN) ranging from 15-35% B (100 mpm) over 40min.

Example 62: Synthesis of Cyclic PYY Analog SEQ ID NO:62

The title compound was prepared following the procedures described inExample 11, substituting 11,11,11-trifluoroundecanoic acid in place ofα-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH˜9)and Buffer B (MeCN) ranging from 10-28% B (100 mpm) over 40 min.

Example 63: Synthesis of Cyclic PYY Analog SEQ ID NO:63

The title compound was prepared following the procedures described inExample 11, substituting 15,15,15-trifluoropentadecanoic acid in placeof α-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH˜9)and Buffer B (MeCN) ranging from 15-30% B (100 mpm) over 40 min.

Example 64: Synthesis of Cyclic PYY Analog SEQ ID NO:64

The title compound was prepared following the procedures described inExample 11, substituting 16-ethoxypalmitic acid in place ofα-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, and usingm-chloromethylbenzoic acid in place of m-bromomethylbenzoic acid in step4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in place ofMeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc buffer instep 4 to effect cyclization. Product purification was performed using aWaters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. The mobile phaseconsisted of a gradient elution of Buffer A (10 mM NH₄OH in water, pH 9)and Buffer B (MeCN) ranging from 15-30% B (100 mpm) over 40 min.

Example 65: Synthesis of Cyclic PYY Analog SEQ ID NO:65

The title compound was prepared following the procedures described inExample 11, substituting 13,13,14,14,15,15,16,16,16-D9-palmitic acid(Cambridge Isotopes) in place of α-Tocopheryloxyacetic Acid (AcVitE) (8)in step 2, and using m-chloromethylbenzoic acid in place ofm-bromomethylbenzoic acid in step 4 (Example 9, step 2). 60% EtOH/H₂Owas used as solvent in place of MeCN/H₂O and sat'd aq. NaHCO₃ was usedin place of the NH₄OAc buffer in step 4 to effect cyclization. Productpurification was performed using a Waters XBridge C18 OBD column (50×250mm, 5 μm) at rt. The mobile phase consisted of a gradient elution ofBuffer A (10 mM NH₄OH in water, pH˜9) and Buffer B (MeCN) ranging from15-20% B (100 mpm) over 5 min, and then to 35% B (100 mpm) over 40 min.

Example 66: Synthesis of Cyclic PYY Analog SEQ ID NO:66

The title compound was prepared following the procedures described inExample 11, substituting 11-[(2,4-bis(trifluoromethyl)phenyl]undecanoicacid in place of α-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, andusing m-chloromethylbenzoic acid in place of m-bromomethylbenzoic acidin step 4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in placeof MeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc bufferin step 4 to effect cyclization. Product purification was performedusing a Waters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. Themobile phase consisted of a gradient elution of Buffer A (10 mM NH₄OH inwater, pH˜9) and Buffer B (MeCN) ranging from 15-35% B (100 mpm) over 40min.

Example 67: Synthesis of Cyclic PYY Analog SEQ ID NO:67

The title compound was prepared following the procedures described inExample 11, substituting 11-[(3,5-bis(trifluoromethyl)phenyl]undecanoicacid in place of α-Tocopheryloxyacetic Acid (AcVitE) (8) in step 2, andusing m-chloromethylbenzoic acid in place of m-bromomethylbenzoic acidin step 4 (Example 9, step 2). 60% EtOH/H₂O was used as solvent in placeof MeCN/H₂O and sat'd aq. NaHCO₃ was used in place of the NH₄OAc bufferin step 4 to effect cyclization. Product purification was performedusing a Waters XBridge C18 OBD column (50×250 mm, 5 μm) at rt. Themobile phase consisted of a gradient elution of Buffer A (10 mM NH₄OH inwater, pH˜9) and Buffer B (MeCN) ranging 15-35% B (100 mpm) over 40 min.

Example 68: Synthesis of Cyclic PYY Analog SEQ ID NO:68 1. Synthesis of(Fmoc)-βA-IKPEAPGEK(Alloc)ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-SieberResin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resinfrom Example 1, step 2 (0.1 mmol) were carried out at rt using DMF assolvent, a 6-fold excess of protected amino acids and an HATU/DIEAprotocol (10 min, double coupling). A two-stage Fmoc deprotectionprotocol was used throughout (20% piperidine in DMF; rt; 10 min, 15min).

2. Synthesis of(Fmoc)-(A-IKPEAPGEK((OEG)₂-γ-Glu-NHCO(CH₂)₁₆CO₂tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-SieberResin

Deprotection of the above resin was carried out following the methoddescribed in Example 1, step 4, using modified reaction times of 10 minfor each treatment. The resin was then coupled with intermediate 2 (15)(5 eq.), using an HATU/DIEA protocol in DMF (1h, rt).

3. Synthesis of(BrAc)-βA-IKPEAPGEK((OEG)₂-γ-Glu-NHCO(CH₂)₁₆CO₂tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-SieberResin

Following Fmoc deprotection (20% piperidine/DMF), the above resin wastreated with bromoacetic anhydride (10 eq.; rt, 30 min) to provide thebromoacetylated resin.

4. Synthesis of(BrAc)-βA-IKPEAPGEK((OEG)₂-γ-Glu-NHCO(CH₂)₁₆CO₂tBu)-ASPEELNRYYASLRHYLNCVTRQ(psi-R35Y36)-CONH₂

The above resin was treated with a cleavage cocktail consisting ofTFA/H₂O/TIPS (95:2.5:2.5) for 1.5 h at rt. The crude peptide wasprecipitated with ether following the procedure described in Example 1,step 7.

5. Cyclic PYY Analog SEQ ID NO: 68

The crude peptide obtained above was dissolved at a concentration of 10mg/mL in 10% MeCN/H₂O, and TEA was added to raise the solution pH to8-9. After stirring at rt for -20 min, TFA was added to lower the pH to2, and the solution was purified directly by preparative HPLC on aKinetics C18 Evo column (30×100 mm, 100 Å, 5 μm). The mobile phaseconsisted of gradient elution of Buffer A (0.1% TFA in water) and BufferB (0.1% TFA in MeCN) ranging from 20-60% B over 22 min. UV detection wasmonitored at 220 and 254 nm. Pure fractions were combined, and thenlyophilized to give the product as a cotton-like solid.

Example 69: Synthesis of Cyclic PYY Analog SEQ ID NO:69 1. Synthesis of(Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(OAllyl)TRQ(psi-R35Y36)-SieberResin

Amino acid extensions onto the pre-loaded (psi-R35, Y36)-Sieber resinfrom Example 1, step 2 (0.1 mmol) were carried out at rt using DMF assolvent, a 6-fold excess of protected amino acids and an HATU/NMMprotocol (10 min, double coupling). A two-stage Fmoc deprotectionprotocol was used throughout (20% piperidine in DMF; rt; 10 min, 15min).

2. Synthesis of(Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(NHS)TRQ(psi-R35Y36)-SieberResin

Alloc-deprotection of the above resin was carried out following themethod described in Example 1, step 4, using modified reaction times of10 min for each treatment. The resin was then coupled with NHS (10 eq.),using an HATU/DIEA protocol in DMF (1 h, rt, double coupling).

3. Synthesis of(NH₂)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLNLE(NHS)TRQ(psi-R35Y36)

The above resin was treated with a cleavage cocktail consisting ofTFA/H₂O/TIPS (95:2.5:2.5) for 1.5 h at rt. The crude peptide wasprecipitated with ether following the procedure described in Example 1,step 7.

4. Cyclic PYY Analog SEQ ID NO: 69

The crude peptide obtained above was dissolved at a concentration of 80mg/mL in DMSO, and TEA (25 eq.) was added to effect lactamization. Afterstirring at rt for -30 min, the reaction was diluted 10-fold with 10%MeCN/water, the pH adjusted to 2 and the crude peptide purified directlyby preparative HPLC on a Kinetics C18 Evo column (30×100 mm, 100 Å, 5μm). The mobile phase consisted of gradient of Buffer A (0.1% TFA inwater) and Buffer B (0.1% TFA in MeCN) ranging from 10-60% B over 22min. UV detection was monitored at 220 and 254 nm. Pure fractions werecombined, and then lyophilized to give the K(Dde)-protected peptide. TheDde protecting group was removed using 2% hydrazine/DMF (10 mgpeptide/ml), 30 mins at rt. The reaction was diluted 10-fold with 10%MeCN/water, and the pH was adjusted to 2 with TFA and the crude peptidesolution was purified as above to give the product as a cotton-likesolid.

Example 70: Synthesis of Cyclic PYY Analog SEQ ID NO:70

The title compound was prepared according to the procedure in Example69, substituting Fmoc-E(OAll)-OH for Fmoc-Leu-OH at position 30 andsubstituting Fmoc-Val-OH for Fmoc-E(OAll)-OH in position 31.

Example 71: Synthesis of Cyclic PYY Analog SEQ ID NO:71 1. Synthesis of(Boc)-G-ISPEAPGEK(dde)ASPEELNRYYASLRHYLN E(OAllyl)VTRQ(N-Me-R)Y-NovaSynTGR Resin

Amino acid extensions onto a NovaSyn TGR resin (0.1 mmol) were carriedout using the procedure described in Example 69, step 1.

2. Cyclic PYY Analog SEQ ID NO: 71

The title compound was prepared from the above resin according to theprocedures described in Example 69, steps 2-4.

Example 72: Synthesis of Cyclic PYY Analog SEQ ID NO:72 1. Synthesis of(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicN-hydroxysuccinimide Ester

To a solution of(S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontan-1-oicacid (Intermediate 2 (16)) (54.0 mg, 0.063 mmol), N-hydroxysuccinimide(14.6 mg, 0.127 mmol), and HATU (24.1 mg, 0.063 mmol) in 1.0 ml of DMFwas added DIEA (0.022 ml, 0.127 mmol) and the mixture stirred for 30mins at RT and used directly in the next step without furtherpurification.

2. Synthesis of Cyclic PYY Analog SEQ ID NO:72

To a solution of [cyclo-(G2-E30), S4, K11, psi-(R35,Y36)]-PYY2-36(prepared in Example 70) (4 mg, 0.96 μmol) in DMF (0.2 mL) was added 24μL of the N-hydroxy ester solution (prepared in Step 1), and TEA (0.66μL; 5 eq) and the mixture stirred overnight at rt. The reaction wasdiluted 10-fold with 10% MeCN/water, the pH adjusted to 2 with TFA andthe crude peptide purified directly by preparative HPLC on a KineticsC18 Evo column (30×100 mm, 100 Å, 5 μm). The mobile phase consisted ofgradient elution of Buffer A (0.1% TFA in water) and Buffer B (0.1% TFAin MeCN) ranging from 10-60% B over 22 min. UV detection was monitoredat 220 and 254 nm. Pure fractions were combined, and then lyophilized togive the t-butyl ester-protected peptide. The t-butyl ester protectinggroups were removed using a mixture of TFA/H₂O/TIPS (95:2.5:2.5) for 1.5h at rt. The mixture was concentrated and the peptide purified as aboveto give the product as a cotton-like solid.

Example 73: Synthesis of Cyclic PYY Analog SEQ ID NO:73

The title compound was prepared according to the procedure as describedin Example 1 substituting N-Fmoc-dPEG6-carboxylic acid forN-Fmoc-dPEG12-carboxylic acid in step 5.

Example 74: Synthesis of Cyclic PYY Analog SEQ ID NO:74

The title compound was prepared according to the procedure as describedin Example 1 but omitting the PEG linker coupling step 5.

Example 75: Synthesis of Cyclic PYY Analog SEQ ID NO:75

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-Cys(trt)-OH for Fmoc-hCys(trt)-OH atposition 31, and omitting the Fmoc-βAla-OH coupling step in step 3.

Example 76: Synthesis of Cyclic PYY Analog SEQ ID NO:76

The title compound was prepared according to the procedure as describedin Example 1 with modified step 3 and step 4. In step 3,Fmoc-Lys(Alloc)-OH and Fmoc-Lys(dde)-OH were used for position 30 andposition 11, respectively. After Alloc at position at 30 was deprotectedwith Pd(PPh₃)₄-phenylsilane, mPEG16-carboxylic acid was coupled withHATU-DIPEA. In step 4, dde at position 11 was removed with 2% hydrazinein DMF.

Example 77: Synthesis of Cyclic PYY Analog SEQ ID NO:77

The title compound was prepared according to the procedure as describedin Example 76 substituting mPEG12-carboxylic acid for mPEG16-carboxylicacid in step 3, and omitting the Fmoc-dPEG12-carboxylic acid couplingstep in step 5.

Example 78: Synthesis of Cyclic PYY Analog SEQ ID NO:78

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-N-Me-Gln(trt)-OH for Fmoc-Gln(trt)-OH instep 3.

Example 79: Synthesis of Cyclic PYY Analog SEQ ID NO:79

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-N-Me-Arg(pbf)-OH for Fmoc-Arg(pbf)-OH instep 1B.

Example 80: Synthesis of Cyclic PYY Analog SEQ ID NO:80

The title compound was prepared according to the procedure as describedin Example 79 substituting Fmoc-Arg(pbf)-OH for Fmoc-Lys(Boc)-OH atposition 4, and substituting Fmoc-Trp(Boc)-OH for Fmoc-Leu-OH atposition 30 in step 3.

Example 81: Synthesis of Cyclic PYY Analog SEQ ID NO:81

The title compound was prepared according to the procedure as describedin Example 80 substituting Fmoc-Cys(trt)-OH for Fmoc-hCys(trt)-OH atposition 31, and substituting Fmoc-γ-aminobutanoic acid for Fmoc-βAla-OHin step 3.

Example 82: Synthesis of Cyclic PYY Analog SEQ ID NO:82

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-PEG2-carboxylic acid for Fmoc-βAla-OH andFmoc-Cys(trt)-OH for Fmoc-hCys(trt)-OH at position 31 as well asomitting the coupling of Fmoc-Ile-OH in step 3.

Example 83: Synthesis of Cyclic PYY Analog SEQ ID NO:83

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-Lys(N3)-OH for Fmoc-hCys(trt)-OH atposition 31, substituting pent-4-ynoic acid for Fmoc-βAla-OH in step 3,and following the cyclization procedure B as below.

Cyclization procedure B: To a solution of fully deprotected peptide withPEG12-AcBr installed at position 11 (38 mg, 0.0067 mmol) in 2 mL ofHEPES (pH 7.4) was added 1.7 mL of the premixed CuSO₄/TBTA solution (thesolution was prepared by mixing a solution of 2.2 mg of CuSO₄ in water(0.4 mL) with a solution of 11 mg of TBTA in EtOH), followed by additionof 7 mg of sodium ascorbate in water (1 mL). The clear reaction solutionwas left mixing at rt and monitored by HPLC. After 30 min, the reactionwas completed, and the reaction mixture was adjusted to pH 4 using TFAand subjected to HPLC purification (Pursuit XRS 5 250×30 mm C18 column,running @ 30 mpm flow, monitoring 214 nM wavelength, with a gradientranging from 20-60% MeCN-water/water both with 0.1% TFA over 36minutes). The desired fraction was collected and lyophilized.

Example 84: Synthesis of Cyclic PYY Analog SEQ ID NO:84

The title compound was prepared according to the procedure as describedin Example 1 omitting the Fmoc-βAla-OH coupling step, substitutingN3-PEG8-carboxylic acid for Fmoc-dPEG12-carboxylic acid in step 5, andsubstituting 3-(bromomethyl)benzoic acid coupling with DIC forbromoacetic anhydride acylation in step 3, and following the cyclizationprocedure C as below.

Cyclization procedure C: To a solution of fully deprotected peptide (20mg, 0.0035 mmol) in 5 mL of degassed water, aq. NaHCO₃ solution wasadded to adjust the reaction mixture to pH 6.4 or higher. After 20 min,the LCMS indicated the reaction was complete, and the reaction mixturewas adjusted to pH 4 using TFA and subjected to HPLC purification(Pursuit XRS 5 250×30 mm C18 column, running @ 30 mpm flow, monitoring214 nM wavelength, with a gradient ranging from 10-60% MeCN-water/waterboth with 0.1% TFA over 36 minutes). The desired fraction was collectedand lyophilized.

After the cyclization, the cyclized intermediate was subjected to linkerextension by click chemistry following the cyclization procedure B withN-(1-bromo-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)pent-4-ynamide,which was prepared by coupling of N-Boc-PEG4-NH₂ with pent-4-ynoic acidusing HATU-DIPEA, followed by deprotection of Boc with TFA and acylationwith bromoacetic anhydride in the presence of TEA.

Example 85: Synthesis of Cyclic PYY Analog SEQ ID NO:85

The title compound was prepared according to the procedure as describedin Example 1 with PEG12-AcBr linker installed at position 23 instead ofposition 11.

Example 86: Synthesis of Cyclic PYY Analog SEQ ID NO:86

The title compound was prepared according to the procedure as describedin Example 1 with PEG12-AcBr linker installed at position 22 instead ofposition 11.

Example 87: Synthesis of Cyclic PYY Analog SEQ ID NO:87

The title compound was prepared according to the procedure as describedin Example 1 with PEG12-AcBr linker installed at position 7 instead ofposition 11.

Example 88: Synthesis of Cyclic PYY Analog SEQ ID NO:88

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-V-OH for Fmoc-hCys(trt)-OH at position31, substituting Fmoc-Cys(trt)-OH for Fmoc-Leu-OH at position 30, andsubstituting Fmoc-Gly-OH for Fmoc-βAla-OH in step 3.

Example 89: Synthesis of Cyclic PYY Analog SEQ ID NO:89

The title compound was prepared according to the procedure as describedin Example 88 omitting step 1 to make the reduced dipeptide, andsubstituting Fmoc-Tyr(tBu)-OH loading followed by coupling withFmoc-(N-Me)Arg-OH for Fmoc-psi-(R35-N(Boc)-Y36)-OH loading in step 2.

Example 90: Synthesis of Cyclic PYY Analog SEQ ID NO:90

The title compound was prepared according to the procedure as describedin Example 89 substituting Fmoc-βAla-OH for Fmoc-Gly-OH in step 3.

Example 91: Synthesis of Cyclic PYY Analog SEQ ID NO:91

The title compound was prepared according to the procedure as describedin Example 89 substituting Fmoc-hCys(trt)-OH for Fmoc-Cys(trt)-OH atposition 30 in step 3.

Example 92: Synthesis of Cyclic PYY Analog SEQ ID NO:92

The title compound was prepared according to the procedure as describedin Example 90 substituting Fmoc-hCys(trt)-OH for Fmoc-Val-OH at position31, and substituting Fmoc-Leu-OH for Fmoc-Cys(trt)-OH at position 30 instep 3.

Example 93: Synthesis of Cyclic PYY Analog SEQ ID NO:93

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-Val-OH for Fmoc-hCys(trt)-OH at position31, substituting Fmoc-Cys(trt)-OH for Fmoc-Leu-OH at position 30, andsubstituting Fmoc-Gly-OH for Fmoc-βAla-OH at the N-terminus in step 3.

Example 94: Synthesis of Cyclic PYY Analog SEQ ID NO:94

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-Val-OH for Fmoc-hCys(trt)-OH at position31 and substituting Fmoc-Cys(trt)-OH for Fmoc-Leu-OH at position 30 instep 3.

Example 95: Synthesis of Cyclic PYY Analog SEQ ID NO:95

The title compound was prepared (0.05 mmol scale) according to theprocedures as described in Example 1 substituting Fmoc-Val-OH forFmoc-hCys(trt)-OH at position 31, substituting Fmoc-Glu(OAlloc)-OH forFmoc-Leu-OH at position 30, substituting Fmoc-Lys(Dde)-OH forFmoc-Lys(Alloc)-OH at position 11, substituting Fmoc-Ser(tBu)-OH forFmoc-Lys(Boc)-OH in position 4 and substituting Boc-Gly-OH forFmoc-βAla-OH at the N-terminus in step 3.

To the resulting resin from above was added deoxygenated DCM (10 mL),phenylsilane (10 eq.) and a solution of the Pd(PPh₃)₄(0.2 eq.) in DCM (1mL) and the mixture was stirred for 10 mins. The reaction was drainedand the resin was washed with deoxygenated DCM and the deprotection wasrepeated one time.

To the resulting resin from above was added DMF (10 ml), HATU (5 eq),and DIEA (10 eq) and the mixture stirred for 5 min then a solution ofN-hydroxysuccinimide (10 eq) in DMF was added and stirred for anadditional 20 min. The resin was filtered and the procedure repeated onetime.

The resin from above was deprotected for 2 h at rt in TFA/TIPS/water(95/2.5/2.5) (10 ml). The cleavage cocktail was concentrated to approx.1 ml and then added to 40 ml of ether. The resulting precipitate wascollected by centrifugation and dried under N2.

The resulting material from above was dissolved in 9 mL of DMSO to which10 eq of TEA was added and the reaction allowed to proceed for 3 h atrt. The resulting solution was diluted to 30 ml with water, the pHadjusted to 2 and purified by RP-HPLC on a 30 mm×250 mm C18 columneluting with a linear gradient of 20-40% MeCN in water (0.1% TFA) in 30mins. The fractions containing product were lyophilized.

The resulting material from above was then treated with 1-2%hydrazine/DMF (1 mL) to remove the Dde from lysine. The resultingmixture was diluted to 10 ml with water, the pH adjusted to 2 and thenpurified by RP-HPLC as above.

The resulting product was then dissolved in 10% MeCN/water, the pHadjusted to 10, and a solution of bromoacetic N-hydroxysuccinimide ester(3 eq of 0.1M/DMF soln) was added and the reaction allowed to proceedfor 10 min at rt. The resulting mixture was diluted to 10 ml with water,the pH adjusted to 2 and then purified by RP-HPLC as above to give thetitle product.

Example 96: Synthesis of Cyclic PYY Analog SEQ ID NO:96

The title compound was prepared according to the procedure as describedin Example 1 substituting N-Fmoc-dPEG24-carboxylic acid forN-Fmoc-dPEG12-carboxylic acid in step 5.

Example 97: Synthesis of Cyclic PYY Analog SEQ ID NO:97

The title compound was prepared according to the procedure as describedin Example 1 substituting Fmoc-Gly-OH for Fmoc-βAla-OH in step 3.

Example 98: Synthesis of Cyclic PYY Analog SEQ ID NO:98

The title compound was prepared according to the procedure as describedin Example 89 but omitting the Fmoc-dPEG12-carboxylic acid coupling stepin step 5.

Example 99: Synthesis of Cyclic PYY Analog SEQ ID NO:99

The title compound was prepared according to the procedure as describedin Example 90 but omitting the Fmoc-dPEG12-carboxylic acid coupling stepin step 5.

Example 100: Synthesis of Cyclic PYY Analog SEQ ID NO:100

The title compound was prepared according to the procedure as describedin Example 94 but omitting the Fmoc-dPEG12-carboxylic acid coupling stepin step 5.

All of the following sequences are considered to be examples of theinvention.

Example 111: Human NPY2R cAMP In Vitro Potency Assay (hY2 Assay)

The method used to test the potency of PYY analogs in vitro was a cellbased assay designed to measure inhibition of forskolin-induced cAMPproduced by adenylate cyclase through modulation of the human NPY2RGi-protein coupled receptor. The forskolin-induced cAMP production inhuman NPY2R transfected HEK cells was reduced through activation ofNPY2R by PYY analogs and controls in a dose-dependent manner, andmeasured in the LANCE FRET-based competitive cAMP immunoassay(PerkinElmer).

Cells were thawed from cryopreservation and added to 15 ml of cell media(DMEM/high glucose (Cellgro), 10% FBS (Hyclone), 1% Pen/Strep (LifeTechnologies), 1% L-Glutamine (Thermo Scientific), 1% Na Pyruvate(Thermo Scientific)). Cells were centrifuged at 450×g for 5 min,supernatants were aspirated, and cells were re-suspended in cell mediaat a density of 0.2×10⁶ cells/ml. Cells were dispensed (25 μL/well) to aBiocoat collagen-coated white 384-well plate (Becton Dickinson) to afinal density of 5000 cells/well, and incubated at 37° C., 5% CO₂ for 16to 24 h. Supernatants in the assay plate were decanted. Dilutions of PYYanalogs and controls were prepared in 1×HBSS (Cellgro), 5 mM HEPES(Cellgro), 0.1% BSA (PerkinElmer) and 0.5 mM 3-isobutyl-1-methylxanthine(Sigma), and 6 μL/well of each sample were added to designated wells.Lance cAMP antibody (PerkinElmer) was diluted 1:100 in 1×HBSS (Cellgro),5 mM HEPES (Cellgro), 0.005 mM forskolin (Sigma), 0.1% BSA (PerkinElmer)and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), and 6 μL of the antibodymixture was added to the plate, which was then incubated at rt for 25min. Then 12 μL/well LANCE cAMP detection reagent mix containingbiotin-cAMP (1:750) and Europium-W8044 (1:2250) (PerkinElmer) was addedto each assay plate, which was then incubated at rt for 2 h. Plates wereread on a PerkinElmer Envision plate reader (excitation 320 nm, emission-615 nm and 665 nm), with relative fluorescence units (RFU) calculatedas (615 nm/665 nm)×10,000. All samples were measured in triplicate. Datawere analyzed using the Crucible in-house data analysis software,designed by Eudean Shaw. The unknown cAMP concentrations within eachwell were interpolated from the reference standards of known cAMPconcentrations included within each plate. Parameters such as ECso,Log(ECso), HillSlope (nH), top, and bottom, were derived by plottingcAMP concentration values over log compound concentrations fitted with4-P model using a non-linear weighted least squares application within Renvironment (Open Source http://cran.us.r-project.org/) implemented bythe Non-Clinical Statistics & Computing department at Janssen R&D.

The potencies of the NTSC-PYY analogues of the present inventionrelative to PYY₃₋₃₆, used as a control in the same assay are presentedin Table 2 below:

TABLE 2 hY2 Receptor Potencies of NTSC-PYY Compounds and PYY₃₋₃₆ (SEQ IDNO: 111) Y2R EC₅₀ Y2R EC₅₀ SEQ ID (nM) (nM) NO. SEQ ID NO: 2-39 SEQ IDNO: 111 2 0.14 0.19 3 0.11 0.12 4 0.74 0.05 5 9.8 0.05 6 10.5 0.05 70.56 0.05 8 0.06 0.05 9 0.02 0.12 10 0.12 0.12 11 0.13 0.15 12 0.21 0.0913 0.04 0.09 14 0.10 0.13 15 0.08 0.12 16 0.17 0.12 17 0.31 0.12 18 1.40.12 19 2.9 0.07 20 4.1 0.07 21 0.49 0.12 22 4.4 0.12 23 9.4 0.12 24 6.10.12 25 0.02 0.12 26 0.03 0.12 27 0.70 0.11 28 0.05 0.11 29 0.09 0.11 301.6 0.08 31 0.01 0.10 32 0.15 0.08 33 0.13 0.08 34 0.09 0.08 35 0.180.10 36 1.5 0.10 37 0.09 0.09 38 0.21 0.09 39 0.02 0.05 40 0.01 0.08 410.07 0.08 42 11.3 0.08 43 0.01 0.09 44 2.11 0.09 45 0.01 0.09 46 0.020.09 47 3.3 0.08 48 6.9 0.10 49 0.35 0.10 50 11.9 0.10 51 0.10 0.10 520.01 0.08 53 0.02 0.08 54 0.09 0.08 55 0.02 0.08 56 0.09 0.06 57 0.030.11 58 0.11 0.07 59 0.01 0.08 60 0.01 0.08 61 0.05 0.08 62 0.12 0.08 630.11 0.06 64 0.08 0.06 65 0.04 0.06 66 0.03 0.06 67 0.07 0.06 68 0.270.06 69 8.97 0.08 70 0.02 0.08 71 0.07 0.04 72 1.75 0.07

Example 112: Efficacy Studies In Vivo

A) Food Intake in Lean C57BL6N Mice: Acute Dosing

Male C57BL/6 mice (10-12 weeks of age) were obtained from TaconicLaboratory. Mice were housed one mouse per cage with AlphaDri bedding ina temperature-controlled room with 12-h light/dark cycle. Mice wereallowed ad libitum access to water and maintained on their regular diet(Lab Diet Cat: 5001). Animals were acclimated in the BioDAQ cages(Research Diets, Inc., New Brunswick, N.J.) no less than 72 h prior tothe start of the experiment.

Once acclimated in the BioDAQ cages, mice were grouped into cohorts ofeight animals based on their individual food intake over the previous 24h. At 4:00-5:00 μm, animals were weighed and treated with either vehicle(2.7 mM disodium phosphate, 61.33 mM propylene glycol, 19.5 mM phenol,pH 8.2) or test compound at a dose of 1 μmol/kg (500 nmol/mL) viasubcutaneous administration. Following compound administration, changesin food weight for each cage were recorded continuously by the BioDAQautomated monitoring system for 24 h. Crumbs were removed daily fromhoppers and the areas around the cages with a vacuum. Food wasreplenished as necessary. The percentage of mean cumulative food intakerelative to vehicle over the 24 h period post compound administrationwas calculated and is reported in Table 3. Statistical analyses wereperformed using one-way ANOVA with Tukey's post-test in Prism. All dataare presented as the mean.

B) Weight Loss in Diet-Induced Obese (DIO) Mice: Acute Dosing

Male DIO C57BL/6 mice (20 weeks of age, 14 weeks on high fat diet) wereobtained from Taconic Laboratory. Mice were housed one mouse per cagewith AlphaDri bedding in a temperature-controlled room with 12-hlight/dark cycle. Mice were allowed ad libitum access to water andmaintained on high fat diet (D12492, Research Diet). Animals wereacclimated to the facility for at least one week prior to the start ofthe experiment.

The day prior to dosing, mice were grouped into cohorts of eight animalsbased on individual body weights. At 3:00-4:00 μm the following day,animals were weighed and treated with either vehicle (2.7 mM disodiumphosphate, 61.33 mM propylene glycol, 19.5 mM phenol, pH 8.2) or testcompound at a dose of 100 nmol/kg (50 nmol/mL) via subcutaneousadministration. Body weights were measured 24 h after dosing and thepercentages of weight loss were calculated and are reported in Table 3.Statistical analyses were performed using one-way ANOVA with Tukey'spost-test in Prism. All data are presented as the mean.

C) Weight Loss in Diet-Induced Obese Mice: Chronic Dosing

Male DIO C57BL/6 mice (20 weeks of age, 14 weeks on high fat diet) wereobtained from Taconic Laboratory. Mice were housed one mouse per cagewith AlphaDri bedding in a temperature-controlled room with 12-hlight/dark cycle. Mice were allowed ad libitum access to water andmaintained on high fat diet (D12492, Research Diet). Animals wereacclimated to the facility for at least one week prior to the start ofthe experiment.

The day prior to dosing, mice were grouped based on individual bodyweights. At 3:00-4:00 μm for each of the next 7 days, animals wereweighed and then treated with either vehicle (2.7 mM disodium phosphate,61.33 mM propylene glycol, 19.5 mM phenol, pH 8.2) or test compound at adose of 100 nmol/kg (50 nmol/mL) via subcutaneous administration. After7 days, body weights were measured and the percentages of weight losswere calculated and are reported in Table 3. Statistical analyses wereperformed using one-way ANOVA with Tukey's post-test in Prism. All dataare presented as the mean.

TABLE 3 In Vivo Efficacy Studies of NTSC-PYY Compounds Food Intake LeanWeight Loss Acute DIO Weight Loss Chronic DIO Mice Mice Mice Seq. I.D. %of Vehicle % Weight Change^(#) (24 h) % Weight Change^(#) (7 days) No.(dose = 1 μM/kg) (dose = 100 nM/kg) (dose = 100 nM/kg) PYY3-36 84   NDND 2 65*** ND ND 3 76*  ND ND 8 48*** −4.95*** −10.23*** 9 83   ND ND 1026*** −2.06**  ND 11 ND −5.10***  −9.65*** 13 ND −3.58*** −10.20*** 14ND −2.56*  −5.66** 25 ND −3.95*** ND 26 ND −4.69*** ND 39 ND −4.73*** ND43 ND −1.96**  ND 46 ND −2.86*  ND 51 ND −4.16*** ND 68 ND −5.68*** NDND = not determined ^(#)% Weight Change relative to vehicle controlanimals *p < 0.05; **p < 0.01; ***p < 0.001

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

All documents cited herein are incorporated by reference.

1. A compound of Formula I:

wherein p is 0 or 1; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3, or 4; q is0 or 1; provided that q is 1 only when Z₃₀ is absent; BRIDGE is-Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —SCH₂C(O)NH—,—(OCH₂CH₂)₂NHC(O)CH₂S, —NHC(O)—, or —CH₂S—; Z₄ is K, A, E, S, or R; Z₇is A or K; Z₉ is G or K; Z₁₁ is D or K; Z₂₂ is A or K; Z₂₃ is S or K;Z₂₆ is A or H; Z₃₀ is L, W, absent, or K; provided that Z₃₀ is absentonly when q is 1; Z₃₄ is

Z₃₅ is

or a derivative thereof; wherein the derivative is the compound ofFormula I that is modified by one or more processes comprisingamidation, glycosylation, carbamylation, sulfation, phosphorylation,cyclization, lipidation, or pegylation; or a pharmaceutically acceptablesalt thereof.
 2. The compound of claim 1, wherein the compound is acompound of Formula I or a compound of Formula I that is modified by oneor more processes comprising amidation, lipidation, or pegylation; or apharmaceutically acceptable salt thereof.
 3. The compound of claim 2,wherein BRIDGE is -Ph-CH₂—S—, -triazolyl-, —NHC(O)CH₂S—, —SCH₂C(O)NH—,—(OCH₂CH₂)₂NHC(O)CH₂S, —NHC(O)—, or —CH₂S—; Z₇ is A or K, wherein theamino side chain of said K is optionally substituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl; Z₉ is G or K, wherein the amino side chain ofsaid K is optionally substituted with

wherein t is 0, 1, or 2; u is 0 or 1; and v is 14, 16, or 18;

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl; Z₁₁ is D or K, wherein the amino side chain ofsaid K is optionally substituted with

wherein w is 0, 1, 2, or 4; x is 0 or 1; and y is 14, 16, or 18;

wherein i is an integer of 0 to 24, and X═Br, I or Cl,

Z₂₂ is A or K, wherein the amino side chain of said K is optionallysubstituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂I, or —C(O)CH₂Cl; Z₂₃ is S or K, wherein the amino side chain ofsaid K is optionally substituted with

wherein i is an integer of 0 to 24, and X═Br, I or Cl, —C(O)CH₂Br,—C(O)CH₂, or —C(O)CH₂Cl; Z₃₀ is L, W, absent, or K, provided that Z₃₀ isabsent only when q is 1, wherein the amino side chain of said K isoptionally substituted with

wherein r is 0, 1, or 2; s is 0 or 1; and q is 14, 16, or 18; or

or a pharmaceutically acceptable salt thereof.
 4. A compound of claim 3wherein m is 0, 1, 2, 3, or 5; n is 1, 2, or 4; Z₇ is A or K, whereinthe amino side chain of said K is substituted with

Z₉ is G or K, wherein the amino side chain of said K is substituted with

wherein t is 0; u is 1; and v is 14; Z₁₁ is D or K, wherein the aminoside chain of said K is optionally substituted with

wherein w is 0, or 4; x is 1; and y is 14;

Z₂₂ is A or K, wherein the amino side chain of said K is substitutedwith

Z₂₃ is S or K, wherein the amino side chain of said K is substitutedwith

Z₃₀ is L, W, absent, or K, provided that Z₃₀ is absent only when q is 1,wherein the amino side chain of said K is substituted with

wherein r is 0, or 2; s is 1; and q is 14, 16, or 18; or

or a pharmaceutically acceptable salt thereof.
 5. A compound of claim 1selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 110, ora pharmaceutically acceptable salt thereof.
 6. A conjugate comprising acompound of claim 1 and a half-life extension moiety conjugated thereto.7. A pharmaceutical composition comprising the compound of claim 1, anda pharmaceutically acceptable carrier.
 8. A method for preventing,treating or ameliorating obesity, comprising administering to a subjectin need thereof an effective amount of the compound of claim 1, or aform, composition or medicament thereof.
 9. A method of preventing,treating or ameliorating a syndrome, disorder or disease, wherein saidsyndrome, disorder or disease is selected from the group consisting ofobesity, type 2 diabetes, metabolic syndrome, insulin resistance anddyslipidemia comprising administering to a subject in need thereof aneffective amount of the compound of claim 1, or a form, composition ormedicament thereof.
 10. The method of claim 9, wherein said syndrome,disorder or disease is type 2 diabetes.
 11. A method of reducing foodintake comprising administering to a subject in need thereof aneffective amount of the compound of claim 1, or a form, composition ormedicament thereof.
 12. A method of modulating Y2 receptor activitycomprising administering to a subject in need thereof an effectiveamount of the compound of claim 1, or a form, composition or medicamentthereof.
 13. A method of treating a disease, disorder or syndromeselected from the group consisting of obesity, type 2 diabetes,metabolic syndrome, insulin resistance and dyslipidemia comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 1, or a form, composition or medicament thereof incombination with at least one antidiabetic agent.
 14. The method ofclaim 13, wherein said antidiabetic agent is a glucagon-like-peptide-1receptor modulator.
 15. A method of preparing a pharmaceuticalcomposition, comprising combining the compound of claim 1, with apharmaceutically acceptable carrier.